Modulators of hedgehog signaling pathways, compositions and uses related thereto

ABSTRACT

The present invention makes available methods and reagents for inhibiting aberrant growth states resulting from hedgehog gain-of-function, ptc loss-of-function or smoothened gain-of-function comprising contacting the cell with a hedgehog antagonist, such as a small molecule, in a sufficient amount to aberrant growth state, e.g., to agonize a normal ptc pathway or antagonize smoothened or hedgehog activity. Such methods and reagents may also inhibit the hedgehog pathway in normal cells, e.g., where normal levels of hedgehog signalling are unwanted.

BACKGROUND OF THE INVENTION

Pattern formation is the activity by which embryonic cells form orderedspatial arrangements of differentiated tissues. The physical complexityof higher organisms arises during embryogenesis through the interplay ofcell-intrinsic lineage and cell-extrinsic signaling. Inductiveinteractions are essential to embryonic patterning in vertebratedevelopment from the earliest establishment of the body plan, to thepatterning of the organ systems, to the generation of diverse cell typesduring tissue differentiation (Davidson, E., (1990) Development 108:365-389; Gurdon, J. B., (1992) Cell 68: 185-199; Jessell, T. M. et al.,(1992) Cell 68: 257-270). The effects of developmental cell interactionsare varied. Typically, responding cells are diverted from one route ofcell differentiation to another by inducing cells that differ from boththe uninduced and induced states of the responding cells (inductions).Sometimes cells induce their neighbors to differentiate like themselves(homeogenetic induction); in other cases a cell inhibits its neighborsfrom differentiating like itself. Cell interactions in early developmentmay be sequential, such that an initial induction between two cell typesleads to a progressive amplification of diversity. Moreover, inductiveinteractions occur not only in embryos, but in adult cells as well, andcan act to establish and maintain morphogenetic patterns as well asinduce differentiation (J. B. Gurdon (1992) Cell 68:185-199).

Members of the Hedgehog family of signaling molecules mediate manyimportant short- and long-range patterning processes during invertebrateand vertebrate development. In the fly, a single hedgehog gene regulatessegmental and imaginal disc patterning. In contrast, in vertebrates, ahedgehog gene family is involved in the control of left-right asymmetry,polarity in the CNS, somites and limb, organogenesis, chondrogenesis andspermatogenesis. The first hedgehog gene was identified by a geneticscreen in the fruitfly Drosophila melanogaster (Nüsslein-Volhard, C. andWieschaus, E. (1980) Nature 287, 795-801). This screen identified anumber of mutations affecting embryonic and larval development. In 1992and 1993, the molecular nature of the Drosophila hedgehog (hh) gene wasreported (C. F., Lee et al. (1992) Cell 71, 33-50), and since then,several hedgehog homologues have been isolated from various vertebratespecies. While only one hedgehog gene has been found in Drosphila andother invertebrates, multiple Hedgehog genes are present in vertebrates.

The vertebrate family of hedgehog genes includes at least four members,e.g., paralogs of the single drosophila hedgehog gene. Exemplaryhedgehog genes and proteins are described in PCT publications WO95/18856 and WO 96/17924. Three of these members, herein referred to asDesert hedgehog (Dhh), Sonic hedgehog (Shh) and Indian hedgehog (Ihh),apparently exist in all vertebrates, including fish, birds, and mammals.A fourth member, herein referred to as tiggie-winkle hedgehog (Thh),appears specific to fish. Desert hedgehog (Dhh) is expressed principallyin the testes, both in mouse embryonic development and in the adultrodent and human; Indian hedgehog (Ihh) is involved in bone developmentduring embryogenesis and in bone formation in the adult; and, Shh, whichas described above, is primarily involved in morphogenic andneuroinductive activities. Given the critical inductive roles ofhedgehog polypeptides in the development and maintenance of vertebrateorgans, the identification of hedghog interacting proteins is ofparamount significance in both clinical and research contexts.

The various Hedgehog proteins consist of a signal peptide, a highlyconserved N-terminal region, and a more divergent C-terminal domain. Inaddition to signal sequence cleavage in the secretory pathway (Lee, J.J. et al. (1992) Cell 71:33-50; Tabata, T. et al. (1992) Genes Dev.2635-2645; Chang, D. E. et al. (1994) Development 120:3339-3353),Hedgehog precursor proteins undergo an internal autoproteolytic cleavagewhich depends on conserved sequences in the C-terminal portion (Lee etal. (1994) Science 266:1528-1537; Porter et al. (1995) Nature374:363-366). This autocleavage leads to a 19 kD N-terminal peptide anda C-terminal peptide of 26-28 kD (Lee et al. (1992) supra; Tabata et al.(1992) supra; Chang et al. (1994) supra; Lee et al. (1994) supra;Bumcrot, D. A., et al. (1995) Mol. Cell. Biol. 15:2294-2303; Porter etal. (1995) supra; Ekker, S. C. et al. (1995) Curr. Biol. 5:944-955; Lai,C. J. et al. (1995) Development 121:2349-2360). The N-terminal peptidestays tightly associated with the surface of cells in which it wassynthesized, while the C-terminal peptide is freely diffusible both invitro and in vivo (Porter et al. (1995) Nature 374:363; Lee et al.(1994) supra; Bumcrot et al. (1995) supra: Mart', E. et al. (1995)Development 121:2537-2547; Roelink, H. et al. (1995) Cell 81:445-455).Interestingly, cell surface retention of the N-terminal peptide isdependent on autocleavage, as a truncated form of HH encoded by an RNAwhich terminates precisely at the normal position of internal cleavageis diffusible in vitro (Porter et al. (1995) supra) and in vivo (Porter,J. A. et al. (1996) Cell 86, 21-34). Biochemical studies have shown thatthe autoproteolytic cleavage of the HH precursor protein proceedsthrough an internal thioester intermediate that subsequently is cleavedin a nucleophilic substitution. As a result, the C-terminal end of theN-peptide is modified by a cholesterol moiety, tethering it to the cellsurface. The biological implications are profound. As a result of thetethering, a high local concentration of N-terminal Hedgehog peptide isgenerated on the surface of the Hedgehog producing cells. It is thisN-terminal peptide which is both necessary and sufficient for short- andlong-range Hedgehog signaling activities in Drosphila and vertebrates(Porter et al. (1995) supra; Ekker et al. (1995) supra; Lai et al.(1995) supra; Roelink, H. et al. (1995) Cell 81:445-455; Porter et al.(1996) supra; Fietz, M. J. et al. (1995) Curr. Biol. 5:643-651; Fan,C.-M. et al. (1995) Cell 81:457465; Mart', E., et al. (1995) Nature375:322-325; Lopez-Martinez et al. (1995) Curr. Biol 5:791-795; Ekker,S. C. et al. (1995) Development 121:2337-2347; Forbes, A. J. et al.(1996) Development 122:1125-1135).

HH has been implicated in short- and long-range patterning processes atvarious sites during Drosphila development. In the establishment ofsegment polarity in early embryos, it has short-range effects whichappear to be directly mediated, while in the patterning of the imaginaldiscs, it induces long range effects both directly and via the inductionof secondary signals.

In vertebrates, several hedgehog genes have been cloned in the past fewyears. Of these genes, Shh has received most of the experimentalattention, as it is expressed in different organizing centers that arethe sources of signals that pattern neighboring tissues. Recent evidenceindicates that Shh is involved in these interactions.

The expression of Shh starts shortly after the onset of gastrulation inthe presumptive midline mesoderm, the node in the mouse (Chang et al.(1994) supra; Echelard, Y. et al. (1993) Cell 75:1417-1430), the rat(Roelink, H. et al. (1994) Cell 76:761-775) and the chick (Riddle, R. D.et al. (1993) Cell 75:1401-1416), and the shield in the zebrafish (Ekkeret al. (1995) supra; Krauss, S. et al.(1993) Cell 75:1431-1444). Inchick embyros, the Shh expression pattern in the node develops aleft-right asymmetry, which appears to be responsible for the left-rightsitus of the heart (Levin, M. et al. (1995) Cell 82:803-814).

In the CNS, Shh from the notochord and the Doorplate appears to induceventral cell fates. When ectopically expressed, Shh leads to aventralization of large regions of the mid- and hindbrain in mouse(Echelard et al. (1993) supra; Goodrich, L. V. et al. (1996) Genes Dev.10:301-312), Xenopus (Roelink, H. et al. (1994) supra; Ruiz i Altaba, A.et al. (1995) Mol. Cell. Neurosci. 6:106-121), and zebrafish (Ekker etal. (1995) supra; Krauss et al. (1993) supra; Hammerschmidt, M., et al.(1996) Genes Dev. 10:647-658). In explants of intermediate neuroectodermat spinal cord levels, Shh protein induces Doorplate and motor neurondevelopment with distinct concentration thresholds, floor plate at highand motor neurons at lower concentrations (Roelink et al. (1995) supra;Mart' et al. (1995) supra; Tanabe, Y. et al. (1995) Curr. Biol.5:651-658). Moreover, antibody blocking suggests that Shh produced bythe notochord is required for notochord-mediated induction of motorneuron fates (Mart' et al. (1995) supra). Thus, high concentration ofShh on the surface of Shh-producing midline cells appears to account forthe contact-mediated induction of Doorplate observed in vitro (Placzek,M. et al. (1993) Development 117:205-218), and the midline positioningof the Doorplate immediately above the notochord in vivo. Lowerconcentrations of Shh released from the notochord and the floorplatepresumably induce motor neurons at more distant ventrolateral regions ina process that has been shown to be contact-independent in vitro(Yamada, T. et al. (1993) Cell 73:673-686). In explants taken atmidbrain and forebrain levels, Shh also induces the appropriateventrolateral neuronal cell types, dopaminergic (Heynes, M. et al.(1995) Neuron 15:35-44; Wang, M. Z. et al. (1995) Nature Med.1:1184-1188) and cholinergic (Ericson, J. et al. (1995) Cell 81:747-756)precursors, respectively, indicating that Shh is a common inducer ofventral specification over the entire length of the CNS. Theseobservations raise a question as to how the differential response to Shhis regulated at particular anteroposterior positions.

Shh from the midline also patterns the paraxial regions of thevertebrate embryo, the somites in the trunk (Fan et al. (1995) supra)and the head mesenchyme rostral of the somites (Hammerschmidt et al.(1996) supra). In chick and mouse paraxial mesoderm explants, Shhpromotes the expression of sclerotome specific markers like Pax1 andTwist, at the expense of the dermamyotomal marker Pax3. Moreover, filterbarrier experiments suggest that Shh mediates the induction of thesclerotome directly rather than by activation of a secondary signalingmechanism (Fan, C.-M. and Tessier-Lavigne, M. (1994) Cell 79,1175-1186).

Shh also includes myotomal gene expression (Hammershmidt et al. (1996)supra; Johnson, R. L. et al. (1994) Cell 79:1165-1173; Münsterberg, A.E. et al. (1995) Genes Dev. 9:2911-2922; Weinberg, E. S. et al. (1996)Development 122:271-280), although recent experiments indicate thatmembers of the WNT family, vertebrate homologues of Drosphila wingless,are required in concert (Münsterberg et al. (1995) supra). Puzzlingly,myotomal induction in chicks requires higher Shh concentrations than theinduction of sclerotomal markers (Münsterberg et al. (1995) supra),although the sclerotome originates from somitic cells positioned muchcloser to the notochord. Similar results were obtained in the zebrafish,where high concentrations of Hedgehog induce myotomal and represssclerotomal marker gene expression (Hammerschmidt et al. (1996) supra).In contrast to amniotes, however, these observations are consistent withthe architecture of the fish embryo, as here, the myotome is thepredominant and more axial component of the somites. Thus, modulation ofShh signaling and the acquisition of new signaling factors may havemodified the somite structure during vertebrate evolution.

In the vertebrate limb buds, a subset of posterior mesenchymal cells,the “Zone of polarizing activity” (ZPA), regulates anteroposterior digitidentity (reviewed in Honig, L. S. (1981) Nature 291:72-73). Ectopicexpression of Shh or application of beads soaked in Shh peptide mimicsthe effect of anterior ZPA grafts, generating a mirror image duplicationof digits (Chang et al. (1994) supra; Lopez-Martinez et al. (1995)supra; Riddle et al. (1993) supra) (FIG. 2g). Thus, digit identityappears to depend primarily on Shh concentration, although it ispossible that other signals may relay this information over thesubstantial distances that appear to be required for AP patterning(100-150 μm). Similar to the interaction of HH and DPP in the Drosphilaimaginal discs, Shh in the vertebrate limb bud activates the expressionof Bmp2 (Francis, P. H. et al. (1994) Development 120:209-218), a dpphomologue. However, unlike DPP in Drosphila, Bmp2 fails to mimic thepolarizing effect of Shh upon ectopic application in the chick limb bud(Francis et al. (1994) supra). In addition to anteroposteriorpatterning, Shh also appears to be involved in the regulation of theproximodistal outgrowth of the limbs by inducing the synthesis of thefibroblast growth factor FGF4 in the posterior apical ectodermal ridge(Laufer, E. et al. (1994) Cell 79:993-1003; Niswander, L. et al.(1994)Nature 371:609-612).

The close relationship between Hedgehog proteins and BMPs is likely tohave been conserved at many, but probably not all sites of vertebrateHedgehog expression. For example, in the chick hindgut, Shh has beenshown to induce the expression of Bmp4, another vertebrate dpp homologue(Roberts, D. J. et al. (1995) Development 121:3163-3174). Furthermore,Shh and Bmp2, 4, or 6 show a striking correlation in their expression inepithelial and mesenchymal cells of the stomach, the urogenital system,the lung, the tooth buds and the hair follicles (Bitgood, M. J. andMcMahon, A. P. (1995) Dev. Biol. 172:126-138). Further, Ihh, one of thetwo other mouse Hedgehog genes, is expressed adjacent to Bmp expressingcells in the gut and developing cartilage (Bitgood and McMahon (1995)supra).

Recent evidence suggests a model in which Ihh plays a crucial role inthe regulation of chondrogenic development (Roberts et al. (1995)supra). During cartilage formation, chondrocytes proceed from aproliferating state via an intermediate, prehypertrophic state todifferentiated hypertrophic chondrocytes. Ihh is expressed in theprehypertrophic chondrocytes and initiates a signaling cascade thatleads to the blockage of chondrocyte differentiation. Its direct targetis the perichondrium around the Ihh expression domain, which responds bythe expression of Gli and Patched (Ptc), conserved transcriptionaltargets of Hedgehog signals (see below). Most likely, this leads tosecondary signaling resulting in the synthesis of parathyroidhormone-related protein (PTHrP) in the periarticular perichondrium.PTHrP itself signals back to the prehypertrophic chondrocytes, blockingtheir further differentiation. At the same time, PTHrP repressesexpression of Ihh, thereby forming a negative feedback loop thatmodulates the rate of chondrocyte differentiation.

Patched was originally identified in Drosphila as a segment polaritygene, one of a group of developmental genes that affect celldifferentiation within the individual segments that occur in ahomologous series along the anterior-posterior axis of the embryo. SeeHooper, J. E. et al. (1989) Cell 59:751; and Nakano, Y. et al. (1989)Nature 341:508. Patterns of expression of the vertebrate homologue ofpatched suggest its involvement in the development of neural tube,skeleton, limbs, craniofacial structure, and skin.

Genetic and functional studies demonstrate that patched is part of thehedgehog signaling cascade, an evolutionarily conserved pathway thatregulates expression of a number of downstream genes. See Perrimon, N.(1995) Cell 80:517; and Perrimon, N. (1996) Cell 86:513. Patchedparticipates in the constitutive transcriptional repression of thetarget genes; its effect is opposed by a secreted glycoprotein, encodedby hedgehog, or a vertebrate homologue, which induces transcriptionalactivation. Genes under control of this pathway include members of theWnt and TGF-beta families.

Patched proteins possess two large extracellular domains, twelvetransmembrane segments, and several cytoplasmic segments. See Hooper,supra; Nakano, supra; Johnson, R. L. et al. (1996) Science 272:1668; andHahn, H. et al. (1996) Cell 85:841. The biochemical role of patched inthe hedgehog signaling pathway is unclear. Direct interaction with thehedgehog protein has, however, been reported (Chen, Y. et al. (1996)Cell 87:553), and patched may participate in a hedgehog receptor complexalong with another transmembrane protein encoded by the smoothened gene.See Perrimon, supra; and Chen, supra. However, other studies have shownpatched appears to act catalytically, and only indirectly suppressessmoothened activity (see, e.g., Taipale et al. (2002) Nature 418,892-896).

The human homologue of patched was recently cloned and mapped tochromosome 9q22.3. See Johnson, supra; and Hahn, supra. This region hasbeen implicated in basal cell nevus syndrome (BCNS), which ischaracterized by developmental abnormalities including rib andcraniofacial alterations, abnormalities of the hands and feet, and spinabifida.

BCNS also predisposes to multiple tumor types, the most frequent beingbasal cell carcinomas (BCC) that occur in many locations on the body andappear within the first two decades of life. Most cases of BCC, however,are unrelated to the syndrome and arise sporadically in small numbers onsun-exposed sites of middle-aged or older people of northern Europeanancestry.

Recent studies in BCNS related and sporadic BCC suggest that afunctional loss of both alleles of patched leads to development of BCC.See Johnson, supra; Hahn, supra; and Gailani, M. R. et al. (1996) NatureGenetics 14:78. Single allele deletions of chromosome 9q22.3 occurfrequently in both sporadic and hereditary BCC. Linkage analysisrevealed that the defective inherited allele was retained and the normalallele was lost in tumors from BCNS patients.

Sporadic tumors also demonstrated a loss of both functional alleles ofpatched. Of twelve tumors in which patched mutations were identifiedwith a single strand conformational polymorphism screening assay, ninehad chromosomal deletion of the second allele and the other three hadinactivating mutations in both alleles (Gailani, supra). The alterationsdid not occur in the corresponding germline DNA.

Most of the identified mutations resulted in premature stop codons orframe shifts. Lench, N. J., et al., Hum. Genet. 1997 October; 100(5-6):497-502. Several, however, were point mutations leading to amino acidsubstitutions in either extracellular or cytoplasmic domains. Thesesites of mutation may indicate functional importance for interactionwith extracellular proteins or with cytoplasmic members of thedownstream signaling pathway.

The involvement of patched in the inhibition of gene expression and theoccurrence of frequent allelic deletions of patched in BCC support atumor suppressor function for this gene. Its role in the regulation ofgene families known to be involved in cell signaling and intercellularcommunication provides a possible mechanism of tumor suppression.

SUMMARY OF THE INVENTION

The present invention makes available methods and reagents forinhibiting activation of the hedgehog signaling pathway, e.g., toinhibit aberrant growth states resulting from phenotypes such as ptcloss-of-function, hedgehog gain-of-function, or smoothenedgain-of-function, comprising contacting the cell with an agent, such asa small molecule, in a sufficient amount to agonize a ptc activity,antagonize a hedgehog activity, or antagonize smoothened activity, e.g.,to reverse or control the aberrant growth state. The present inventioncontemplates hedgehog antagonists that inhibit the hedgehog pathway in asmoothened gain-of-function cell with an IC₅₀ within a factor of 20, 10,or even a factor of 5, of the IC₅₀ with which they inhibit the hedgehogpathway in a patched-null cell.

DETAILED DESCRIPTION OF THE INVENTION

I. Overview

The present invention relates to the discovery that signal transductionpathways regulated by hedgehog, patched (ptc), gli and/or smoothened canbe inhibited, at least in part, by small molecules. While not wishing tobe bound by any particular theory, the activation of a receptor may bethe mechanism by which these agents act. For example, the ability ofthese agents to inhibit proliferation of patched loss-of-function(ptc^(lof)) cells may be due to the ability of such molecules tointeract with hedgehog, patched, or smoothened, or at least to interferewith the ability of those proteins to activate a hedgehog, ptc, and/orsmoothened-mediated signal transduction pathway. Experiments relating tosmall molecule inhibitors of the hedgehog pathway are discussed in Chenet al., Proc. Nat. Acad. Sci. 2002, 99:22, 14071-14076; Taipale et al.,Nature 2002, 418, 892-897; and Taipale et al., Nature 2000, 406,1005-1009, all of which are incorporated herein by reference in theirentirety. These studies suggest that hedgehog antagonists of differentstructures, even ones that bind to the same protein in the hedgehogpathway, may act in slightly different ways. Accordingly, even if aparticular condition caused or contributed to by aberrant or unwantedactivation of the hedgehog pathway shows little response to treatment byone of the antagonists disclosed herein, another of the antagonistsdisclosed herein may nonetheless be efficacious.

It is, therefore, specifically contemplated that these small moleculeswhich intefere with aspects of hedgehog, ptc, or smoothened signaltransduction activity will likewise be capable of inhibitingproliferation (or other biological consequences) in normal cells and/orcells having a patched loss-of-function phenotype, a hedgehoggain-of-function phenotype, or a smoothened gain-of-function phenotype.Thus, it is contemplated that in certain embodiments, these compoundsmay be useful for inhibiting hedgehog activity in normal cells, e.g.,which do not have a genetic mutation that activates the hedgehogpathway. In preferred embodiments, the subject inhibitors are organicmolecules having a molecular weight less than 2500 amu, more preferablyless than 1500 amu, and even more preferably less than 750 amu, and arecapable of inhibiting at least some of the biological activities ofhedgehog proteins, preferably specifically in target cells.

Thus, the methods of the present invention include the use of smallmolecules which agonize ptc inhibition of hedgehog signalling, such asby inhibiting activation of smoothened or downstream components of thesignal pathway, in the regulation of repair and/or functionalperformance of a wide range of cells, tissues and organs, includingnormal cells, tissues, and organs, as well as those having the phenotypeof ptc loss-of-function, hedgehog gain-of-function, or smoothenedgain-of-function. For instance, the subject method has therapeutic andcosmetic applications ranging from regulation of neural tissues, boneand cartilage formation and repair, regulation of spermatogenesis,regulation of smooth muscle, regulation of lung, liver and other organsarising from the primitive gut, regulation of hematopoietic function,regulation of skin and hair growth, etc. Moreover, the subject methodscan be performed on cells that are provided in culture (in vitro), or oncells in a whole animal (in vivo). See, for example, PCT publications WO95/18856 and WO 96/17924 (the specifications of which are expresslyincorporated by reference herein).

In a preferred embodiment, the subject method can be to treat epithelialcells having a phenotype of ptc loss-of-function, hedgehoggain-of-function, or smoothened gain-of-function. For instance, thesubject method can be used in treating or preventing basal cellcarcinoma or other hedgehog pathway-related disorders.

In certain embodiments, a subject antagonist may inhibit activation of ahedgehog pathway by binding to smoothened.

In another preferred embodiment, the subject method can be used as partof a treatment regimen for one or more of: malignant medulloblastoma andother primary CNS malignant neuroectodermal tumors (Berman et al.Science, 2002), rhabdomyosarcoma, lung cancer (in particular, small celllung cancer), gut-derived tumors (including but not limited to cancer ofthe esophagus, stomach, pancreas, and biliary duct system), andprostatic and bladder cancers.

In another aspect, the present invention provides pharmaceuticalpreparations comprising, as an active ingredient, a hedgehog antagonist,ptc agonist, or smoothened antagonist such as described herein,formulated in an amount sufficient to inhibit, in vivo, proliferation orother biological consequences of ptc loss-of-function, hedgehoggain-of-function, or smoothened gain-of-function.

The subject treatments using hedgehog antagonists, patched agonists, orsmoothened antagonists can be effective for both human and animalsubjects. Animal subjects to which the invention is applicable extend toboth domestic animals and livestock, raised either as pets or forcommercial purposes. Examples are dogs, cats, cattle, horses, sheep,hogs, and goats.

II. Definitions

For convience, certain terms employed in the specification, examples,and appended claims are collected here.

The phrase “aberrant modification or mutation” of a gene refers to suchgenetic lesions as, for example, deletions, substitution or addition ofnucleotides to a gene, as well as gross chromosornal rearrangements ofthe gene and/or abnormal methylation of the gene. Likewise,mis-expression of a gene refers to aberrant levels of transcription ofthe gene relative to those levels in a normal cell under similarconditions, as well as non-wild-type splicing of mRNA transcribed fromthe gene.

“Basal cell carcinomas” exist in a variety of clinical and histologicalforms such as nodular-ulcerative, superficial, pigmented, morphealike,fibroepithelioma and nevoid syndrome. Basal cell carcinomas are the mostcommon cutaneous neoplasms found in humans. The majority of new cases ofnonmelanoma skin cancers fall into this category.

“Burn wounds” refer to cases where large surface areas of skin have beenremoved or lost from an individual due to heat and/or chemical agents.

The term “carcinoma” refers to a malignant new growth made up ofepithelial cells tending to infiltrate surrounding tissues and to giverise to metastases. Exemplary carcinomas include: “basal cellcarcinoma”, which is an epithelial tumor of the skin that, while seldommetastasizing, has potentialities for local invasion and destruction;“squamous cell carcinoma”, which refers to carcinomas arising fromsquamous epithelium and having cuboid cells; “carcinosarcoma”, whichinclude malignant tumors composed of carcinomatous and sarcomatoustissues; “adenocystic carcinoma”, carcinoma marked by cylinders or bandsof hyaline or mucinous stroma separated or surrounded by nests or cordsof small epithelial cells, occurring in the mammary and salivary glands,and mucous glands of the respiratory tract; “epidermoid carcinoma”,which refers to cancerous cells which tend to differentiate in the sameway as those of the epidermis; i.e., they tend to form prickle cells andundergo cornification; “nasopharyngeal carcinoma”, which refers to amalignant tumor arising in the epithelial lining of the space behind thenose; and “renal cell carcinoma”, which pertains to carcinoma of therenal parenchyma composed of tubular cells in varying arrangements.Other carcinomatous epithelial growths are “papillomas”, which refers tobenign tumors derived from epithelium and having a papillomavirus as acausative agent; and “epidermoidomas”, which refers to a cerebral ormeningeal tumor formed by inclusion of ectodermal elements at the timeof closure of the neural groove.

The “corium” or “dermis” refers to the layer of the skin deep to theepidermis, consisting of a dense bed of vascular connective tissue, andcontaining the nerves and terminal organs of sensation. The hair roots,and sebaceous and sweat glands are structures of the epidermis which aredeeply embedded in the dermis.

“Dental tissue” refers to tissue in the mouth that is similar toepithelial tissue, for example gum tissue. The method of the presentinvention is useful for treating periodontal disease.

“Dermal skin ulcers” refer to lesions on the skin caused by superficialloss of tissue, usually with inflammation. Dermal skin ulcers that canbe treated by the method of the present invention include decubitusulcers, diabetic ulcers, venous stasis ulcers and arterial ulcers.Decubitus wounds refer to chronic ulcers that result from pressureapplied to areas of the skin for extended periods of time. Wounds ofthis type are often called bedsores or pressure sores. Venous stasisulcers result from the stagnation of blood or other fluids fromdefective veins. Arterial ulcers refer to necrotic skin in the areaaround arteries having poor blood flow.

The term “ED₅₀” means the dose of a drug that produces 50% of itsmaximum response or effect.

An “effective amount” of, e.g., a hedgehog antagonist, with respect tothe subject method of treatment, refers to an amount of the antagonistin a preparation which, when applied as part of a desired dosage regimenbrings about, e.g., a change in the rate of cell proliferation and/orthe state of differentiation of a cell and/or rate of survival of a cellaccording to clinically acceptable standards for the disorder to betreated or the cosmetic purpose.

The terms “epithelia”, “epithelial” and “epithelium” refer to thecellular covering of internal and external body surfaces (cutaneous,mucous and serous), including the glands and other structures derivedtherefrom, e.g., corneal, esophegeal, epidermal, and hair follicleepithelial cells. Other exemplary epithlelial tissue includes: olfactoryepithelium, which is the pseudostratified epithelium lining theolfactory region of the nasal cavity, and containing the receptors forthe sense of smell; glandular epithelium, which refers to epitheliumcomposed of secreting cells; squamous epithelium, which refers toepithelium composed of flattened plate-like cells. The term epitheliumcan also refer to transitional epithelium, like that which ischaracteristically found lining hollow organs that are subject to greatmechanical change due to contraction and distention, e.g., tissue whichrepresents a transition between stratified squamous and columnarepithelium.

The term “epithelialization” refers to healing by the growth ofepithelial tissue over a denuded surface.

The term “epidermal gland” refers to an aggregation of cells associatedwith the epidermis and specialized to secrete or excrete materials notrelated to their ordinary metabolic needs. For example, “sebaceousglands” are holocrine glands in the corium that secrete an oilysubstance and sebum. The term “sweat glands” refers to glands thatsecrete sweat, situated in the corium or subcutaneous tissue, opening bya duct on the body surface.

The term “epidermis” refers to the outermost and nonvascular layer ofthe skin, derived from the embryonic ectoderm, varying in thickness from0.07-1.4 mm. On the palmar and plantar surfaces it comprises, fromwithin outward, five layers: basal layer composed of columnar cellsarranged perpendicularly; prickle-cell or spinous layer composed offlattened polyhedral cells with short processes or spines; granularlayer composed of flattened granular cells; clear layer composed ofseveral layers of clear, transparent cells in which the nuclei areindistinct or absent; and horny layer composed of flattened, cornifiednon-nucleated cells. In the epidermis of the general body surface, theclear layer is usually absent.

“Excisional wounds” include tears, abrasions, cuts, punctures orlacerations in the epithelial layer of the skin and may extend into thedermal layer and even into subcutaneous fat and beyond. Excisionalwounds can result from surgical procedures or from accidentalpenetration of the skin.

The “growth state” of a cell refers to the rate of proliferation of thecell and/or the state of differentiation of the cell. An “altered growthstate” is a growth state characterized by an abnormal rate ofproliferation, e.g., a cell exhibiting an increased or decreased rate ofproliferation relative to a normal cell.

The term “hair” refers to a threadlike structure, especially thespecialized epidermal structure composed of keratin and developing froma papilla sunk in the corium, produced only by mammals andcharacteristic of that group of animals. Also, “hair” may refer to theaggregate of such hairs. A “hair follicle” refers to one of thetubular-invaginations of the epidermis enclosing the hairs, and fromwhich the hairs grow. “Hair follicle epithelial cells” refers toepithelial cells that surround the dermal papilla in the hair follicle,e.g., stem cells, outer root sheath cells, matrix cells, and inner rootsheath cells. Such cells may be normal non-malignant cells, ortransformed/immortalized cells.

The term “hedgehog antagonist” refers to an agent that inhibts hedgehogsignalling, such as to repress transcription of target genes or regulateinteractions among proteins and/or signalling molecules. Preferredhedgehog antagonists can be used to overcome a ptc loss-of-functionand/or a smoothened gain-of-function, the latter also being refered toas smoothened antagonists. The term ‘hedgehog antagonist’ as used hereinrefers not only to any agent that may act by directly inhibiting thenormal function of the hedgehog protein, but also to any agent thatinhibits the hedgehog signalling pathway at any level, e.g.,recapitulates the function of ptc or blocks the signalling pathway at adownstream location.

The term “hedgehog gain-of-function” refers to an aberrant modificationor mutation of a ptc gene, hedgehog gene, or smoothened gene, or adecrease (or loss) in the level of expression of such a gene, whichresults in a phenotype which resembles contacting a cell with a hedgehogprotein, e.g., aberrant activation of a hedgehog pathway. Thegain-of-function may include a loss of the ability of the ptc geneproduct to regulate the level of expression of Ci genes, e.g., Gli1,Gli2, and Gli3. The term ‘hedgehog gain-of-function’ is also used hereinto refer to any similar cellular phenotype (e.g., exhibiting excessproliferation) that occurs due to an alteration anywhere in the hedgehogsignal transduction pathway, including, but not limited to, amodification or mutation of hedgehog itself. For example, a tumor cellwith an abnormally high proliferation rate due to activation of thehedgehog signalling pathway would have a ‘hedgehog gain-of-function’phenotype, even if hedgehog is not mutated in that cell.

As used herein, “immortalized cells” refers to cells that have beenaltered via chemical and/or recombinant means such that the cells havethe ability to grow through an indefinite number of divisions inculture.

“Internal epithelial tissue” refers to tissue inside the body that hascharacteristics similar to the epidermal layer in the skin. Examplesinclude the lining of the intestine. The method of the present inventionis useful for promoting the healing of certain internal wounds, forexample wounds resulting from surgery.

The term “IC₅₀” means the concentration of a drug which inhibits anactivity by 50%, e.g., by reducing the frequency of a condition, such ascell death, by 50%, or by reducing the level of an activity, such asproliferation or a level of biological signalling, by 50%.

The term “keratosis” refers to proliferative skin disorder characterizedby hyperplasia of the horny layer of the epidermis. Exemplary keratoticdisorders include keratosis follicularis, keratosis palmaris etplantaris, keratosis pharyngea, keratosis pilaris, and actinickeratosis.

The term “LD₅₀” means the dose of a drug that is lethal in 50% of testsubjects.

The term “nail” refers to the horny cutaneous plate on the dorsalsurface of the distal end of a finger or toe.

The term “patched loss-of-function” refers to an aberrant modificationor mutation of a ptc gene, or a decreased level of expression of thegene, which results in a phenotype which resembles contacting a cellwith a hedgehog protein, e.g., aberrant activation of a hedgehogpathway. The loss-of-function may include a loss of the ability of theptc gene product to regulate the level of expression or activity of Cigenes, e.g., Gli1, Gli2 and Gli3. The term ‘ptc loss-of-function’ isalso used herein to refer to any similar cellular phenotype (e.g.,exhibiting excess proliferation) that occurs due to an alterationanywhere in the hedgehog signal transduction pathway, including, but notlimited to, a modification or mutation of ptc itself. For example, atumor cell with an abnormally high proliferation rate due to activationof the hedgehog signalling pathway would have a ‘ptc loss-of-function’phenotype, even if ptc is not mutated in that cell.

A “patient” or “subject” to be treated by the subject method can meaneither a human or non-human animal.

The term “preventing” is art recognized and when used in relation to acondition, such as a local recurrence (e.g., peripheral neuropathy), adisease such as cancer, a syndrome complex such as BCNS, or any othermedical condition, is well understood in the art, and includesadministration of a composition which reduces the frequency of, ordelays the onset of, symptoms of a medical condition in a subjectrelative to a subject which does not receive the composition. Thus,prevention of cancer includes, for example, reducing the number ofdetectable cancerous growths in a population of patients receiving aprophylactic treatment relative to an untreated control population,and/or delaying the appearance of detectable cancerous growths in atreated population versus an untreated control population, e.g., by astatistically and/or clinically significant amount. Prevention ofperipheral neuropathy includes, for example, reducing the number ofdiagnoses of the infection in a treated population versus an untreatedcontrol population, and/or delaying the onset of symptoms of theneuropathy in a treated population versus an untreated controlpopulation. Prevention of BCNS includes, for example, reducing theseverity of, or alternatively delaying, the formation of growths insubjects in a treated population versus an untreated control population.

The term “prodrug” is intended to encompass compounds that, underphysiological conditions, are converted into the therapeutically activeagents of the present invention. A common method for making a prodrug isto include selected moieties that are hydrolyzed under physiologicalconditions to reveal the desired molecule. In other embodiments, theprodrug is converted by an enzymatic activity of the host animal.

As used herein, “proliferating” and “proliferation” refer to cellsundergoing mitosis.

The term “preventing” is art-recognized, and when used in relation to acondition, such as a local recurrence (e.g., pain), a disease such ascancer, a syndrome complex such as heart failure or any other medicalcondition, is well understood in the art, and includes administration ofa composition which reduces the frequency of, or delays the onset of,symptoms of a medical condition in a subject relative to a subject whichdoes not receive the composition. Thus, prevention of cancer includes,for example, reducing the number of detectable cancerous growths in apopulation of patients receiving a prophylactic treatment relative to anuntreated control population, and/or delaying the appearance ofdetectable cancerous growths in a treated population versus an untreatedcontrol population, e.g., by a statistically and/or clinicallysignificant amount. Prevention of an infection includes, for example,reducing the number of diagnoses of the infection in a treatedpopulation versus an untreated control population, and/or delaying theonset of symptoms of the infection in a treated population versus anuntreated control population. Prevention of pain includes, for example,reducing the magnitude of, or alternatively delaying, pain sensationssubsequently experienced by subjects in a treated population versus anuntreated control population.

Throughout this application, the term “proliferative skin disorder”refers to any disease/disorder of the skin marked by unwanted oraberrant proliferation of cutaneous tissue. These conditions aretypically characterized by epidermal cell proliferation or incompletecell differentiation, and include, for example, X-linked ichthyosis,psoriasis, atopic dermatitis, allergic contact dermatitis, epidermolytichyperkeratosis, and seborrheic dermatitis. For example,epidermodysplasia is a form of faulty development of the epidermis.Another example is “epidermolysis”, which refers to a loosened state ofthe epidermis with formation of blebs and bullae either spontaneously orat the site of trauma.

As used herein, the term “psoriasis” refers to a hyperproliferative skindisorder that alters the skin's regulatory mechanisms. In particular,lesions are formed which involve primary and secondary alterations inepidermal proliferation, inflammatory responses of the skin, and anexpression of regulatory molecules such as lymphokines and inflammatoryfactors. Psoriatic skin is morphologically characterized by an increasedturnover of epidermal cells, thickened epidermis, abnormalkeratinization, inflammatory cell infiltrates into the dermis layer andpolymorphonuclear leukocyte infiltration into the epidermis layerresulting in an increase in the basal cell cycle. Additionally,hyperkeratotic and parakeratotic cells are present.

The term “skin” refers to the outer protective covering of the body,consisting of the corium and the epidermis, and is understood to includesweat and sebaceous glands, as well as hair follicle structures.Throughout the present application, the adjective “cutaneous” may beused, and should be understood to refer generally to attributes of theskin, as appropriate to the context in which they are used.

The term “smoothened gain-of-function” refers to an aberrantmodification or mutation of a smo gene, or an increased level ofexpression of the gene, which results in a phenotype that resemblescontacting a cell with a hedgehog protein, e.g., aberrant activation ofa hedgehog pathway. While not wishing to be bound by any particulartheory, it is noted that ptc may not signal directly into the cell, butrather modulates the activity of smoothened, another membrane-boundprotein located downstream of ptc in hedgehog signaling (Marigo et al.,(1996) Nature 384: 177-179; Taipale et al. (2002) Nature 418, 892-896).The gene smo is a segment-polarity gene required for the correctpatterning of every segment in Drosphila (Alcedo et al., (1996) Cell 86:221-232). Human homologs of smo have been identified. See, for example,Stone et al. (1996) Nature 384:129-134, and GenBank accession U84401.The smoothened gene encodes an integral membrane protein withcharacteristics of heterotrimeric G-protein-coupled receptors; i.e.,7-transmembrane regions. This protein shows homology to the DrosphilaFrizzled (Fz) protein, a member of the wingless pathway. It wasoriginally thought that smo encodes a receptor of the Hh signal.However, this suggestion was subsequently disproved, as evidence for ptcbeing the Hh receptor was obtained. Cells that express Smo fail to bindHh, indicating that smo does not interact directly with Hh (Nusse,(1996) Nature 384: 119-120). Rather, the binding of Sonic hedgehog (SHH)to its receptor, PTCH, is thought to prevent normal inhibition by PTCHof smoothened (SMO), a seven-span transmembrane protein.

Recently, it has been reported that activating smoothened mutationsoccur in sporadic basal cell carcinoma, Xie et al. (1998) Nature 391:90-2, and primitive neuroectodermal tumors of the central nervoussystem, Reifenberger et al. (1998) Cancer Res 58: 1798-803.

The term “therapeutic index” refers to the therapeutic index of a drugdefined as LD₅₀/ED₅₀.

As used herein, “transformed cells” refers to cells that havespontaneously converted to a state of unrestrained growth, i.e., theyhave acquired the ability to grow through an indefinite number ofdivisions in culture. Transformed cells may be characterized by suchterms as neoplastic, anaplastic and/or hyperplastic, with respect totheir loss of growth control.

The term “acylamino” is art-recognized and refers to a moiety that canbe represented by the general formula:

wherein R₉ is as defined above, and R′₁₁ represents a hydrogen, analkyl, an alkenyl or —(CH₂)_(m)—R₈, where m and R₈ are as defined above.

Herein, the term “aliphatic group” refers to a straight-chain,branched-chain, or cyclic aliphatic hydrocarbon group and includessaturated and unsaturated aliphatic groups, such as an alkyl group, analkenyl group, and an alkynyl group.

The terms “alkenyl” and “alkynyl” refer to unsaturated aliphatic groupsanalogous in length and possible substitution to the alkyls describedabove, but that contain at least one double or triple bond respectively.

The terms “alkoxyl” or “alkoxy” as used herein refers to an alkyl group,as defined above, having an oxygen radical attached thereto.Representative alkoxyl groups include methoxy, ethoxy, propyloxy,tert-butoxy and the like. An “ether” is two hydrocarbons covalentlylinked by an oxygen. Accordingly, the substituent of an alkyl thatrenders that alkyl an ether is or resembles an alkoxyl, such as can berepresented by one of —O-alkyl, —O-alkenyl, —O-alkynyl, —O—(CH₂)_(m)—R₈,where m and R₈ are described above.

The term “alkyl” refers to the radical of saturated aliphatic groups,including straight-chain alkyl groups, branched-chain alkyl groups,cycloalkyl (alicyclic) groups, alkyl-substituted cycloalkyl groups, andcycloalkyl-substituted alkyl groups. In preferred embodiments, astraight chain or branched chain alkyl has 30 or fewer carbon atoms inits backbone (e.g., C₁-C₃₀ for straight chains, C₃-C₃₀ for branchedchains), and more preferably 20 or fewer. Likewise, preferredcycloalkyls have from 3-10 carbon atoms in their ring structure, andmore preferably have 5, 6 or 7 carbons in the ring structure.

Moreover, the term “alkyl” (or “lower alkyl”) as used throughout thespecification, examples, and claims is intended to include both“unsubstituted alkyls” and “substituted alkyls”, the latter of whichrefers to alkyl moieties having substituents replacing a hydrogen on oneor more carbons of the hydrocarbon backbone. Such substituents caninclude, for example, a halogen, a hydroxyl, a carbonyl (such as acarboxyl, an alkoxycarbonyl, a formyl, or an acyl), a thiocarbonyl (suchas a thioester, a thioacetate, or a thioformate), an alkoxyl, aphosphoryl, a phosphate, a phosphonate, a phosphinate, an amino, anamido, an amidine, an imine, a cyano, a nitro, an azido, a sulfhydryl,an alkylthio, a sulfate, a sulfonate, a sulfamoyl, a sulfonamido, asulfonyl, a heterocyclyl, an aralkyl, or an aromatic or heteroaromaticmoiety. It will be understood by those skilled in the art that themoieties substituted on the hydrocarbon chain can themselves besubstituted, if appropriate. For instance, the substituents of asubstituted alkyl may include substituted and unsubstituted forms ofamino, azido, imino, amido, phosphoryl (including phosphonate andphosphinate), sulfonyl (including sulfate, sulfonamido, sulfamoyl andsulfonate), and silyl groups, as well as ethers, alkylthios, carbonyls(including ketones, aldehydes, carboxylates, and esters), —CF₃, —CN andthe like. Exemplary substituted alkyls are described below. Cycloalkylscan be further substituted with alkyls, alkenyls, alkoxys, alkylthios,aminoalkyls, carbonyl-substituted alkyls, —CF₃, —CN, and the like.

Unless the number of carbons is otherwise specified, “lower alkyl” asused herein means an alkyl group, as defined above, but having from oneto ten carbons, more preferably from one to six carbon atoms in itsbackbone structure. Likewise, “lower alkenyl” and “lower alkynyl” havesimilar chain lengths. Throughout the application, preferred alkylgroups are lower alkyls. In preferred embodiments, a substituentdesignated herein as alkyl is a lower alkyl.

The term “alkylthio” refers to an alkyl group, as defined above, havinga sulfur radical attached thereto. In preferred embodiments, the“alkylthio” moiety is represented by one of —S-alkyl, —S-alkenyl,—S-alkynyl, and —S—(CH₂)_(m)—R₈, wherein m and R₈ are defined above.Representative alkylthio groups include methylthio, ethylthio, and thelike.

The terms “amine” and “amino” are art-recognized and refer to bothunsubstituted and substituted amines, e.g., a moiety that can berepresented by the general formula:

wherein R₉, R₁₀ and R′₁₀ each independently represent a hydrogen, analkyl, an alkenyl, —(CH₂)_(m)—R₈, or R₉ and R₁₀ taken together with theN atom to which they are attached complete a heterocycle having from 4to 8 atoms in the ring structure; R₈ represents an aryl, a cycloalkyl, acycloalkenyl, a heterocycle or a polycycle; and m is zero or an integerin the range of 1 to 8. In preferred embodiments, only one of R₉ or R₁₀can be a carbonyl, e.g., R₉, R₁₀ and the nitrogen together do not forman imide. In certain such embodiments, neither R₉ and R₁₀ is attached toN by a carbonyl, e.g., the amine is not an amide or imide, and the amineis preferably basic, e.g., its conjugate acid has a pK_(a) above 7. Ineven more preferred embodiments, R₉ and R₁₀ (and optionaly R′₁₀) eachindependently represent a hydrogen, an alkyl, an alkenyl, or—(CH₂)_(m)—R₈. Thus, the term “alkylamine” as used herein means an aminegroup, as defined above, having a substituted or unsubstituted alkylattached thereto, i.e., at least one of R₉ and R₁₀ is an alkyl group.

The term “amido” is art-recognized as an amino-substituted carbonyl andincludes a moiety that can be represented by the general formula:

wherein R₉, R₁₀ are as defined above. Preferred embodiments of the amidewill not include imides that may be unstable.

The term “aralkyl”, as used herein, refers to an alkyl group substitutedwith an aryl group (e.g., an aromatic or heteroaromatic group).

The term “aryl” as used herein includes 5-, 6-, and 7-memberedsingle-ring aromatic groups that may include from zero to fourheteroatoms, for example, benzene, pyrrole, furan, thiophene, imidazole,oxazole, thiazole, triazole, pyrazole, pyridine, pyrazine, pyridazineand pyrimidine, and the like. Those aryl groups having heteroatoms inthe ring structure may also be referred to as “aryl heterocycles” or“heteroaromatics.” The aromatic ring can be substituted at one or morering positions with such substituents as described above, for example,halogen, azide, alkyl, aralkyl, alkenyl, alkynyl, cycloalkyl, hydroxyl,alkoxyl, amino, nitro, sulfhydryl, imino, amido, phosphate, phosphonate,phosphinate, carbonyl, carboxyl, silyl, ether, alkylthio, sulfonyl,sulfonamido, ketone, aldehyde, ester, heterocyclyl, aromatic orheteroaromatic moieties, —CF₃, —CN, or the like. The term “aryl” alsoincludes polycyclic ring systems having two or more cyclic rings inwhich two or more carbons are common to two adjoining rings (the ringsare “fused rings”) wherein at least one of the rings is aromatic, e.g.,the other cyclic rings can be cycloalkyls, cycloalkenyls, cycloalkynyls,aryls and/or heterocyclyls.

The term “carbocycle”, as used herein, refers to an aromatic ornon-aromatic ring in which each atom of the ring is carbon.

The term “carbonyl” is art-recognized and includes such moieties as canbe represented by the general formula:

wherein X is a bond or represents an oxygen or a sulfur, and R₁₁represents a hydrogen, an alkyl, an alkenyl, —(CH₂)_(m)—R₈ or apharmaceutically acceptable salt, R′₁₁ represents a hydrogen, an alkyl,an alkenyl or —(CH₂)_(m)—R₈, where m and R₈ are as defined above. WhereX is an oxygen and R₁₁ or R′₁₁ is not hydrogen, the formula representsan “ester”. Where X is an oxygen, and R₁₁ is as defined above, themoiety is referred to herein as a carboxyl group, and particularly whenR₁₁ is a hydrogen, the formula represents a “carboxylic acid”. Where Xis an oxygen, and R′₁₁ is hydrogen, the formula represents a “formate”.In general, where the oxygen atom of the above formula is replaced bysulfur, the formula represents a “thiocarbonyl” group. Where X is asulfur and R₁₁ or R′₁₁ is not hydrogen, the formula represents a“thioester.” Where X is a sulfur and R₁₁ is hydrogen, the formularepresents a “thiocarboxylic acid.” Where X is a sulfur and R₁₁′ ishydrogen, the formula represents a “thiolformate.” On the other hand,where X is a bond, and R₁₁ is not hydrogen, the above formula representsa “ketone” group. Where X is a bond, and R₁₁ is hydrogen, the aboveformula represents an “aldehyde” group.

The term “heteroatom” as used herein means an atom of any element otherthan carbon or hydrogen. Preferred heteroatoms are boron, nitrogen,oxygen, phosphorus, sulfur and selenium.

The terms “heterocyclyl” or “heterocyclic group” refer to 3- to10-membered ring structures, more preferably 3- to 7-membered rings,whose ring structures include one to four heteroatoms. Heterocycles canalso be polycycles. Heterocyclyl groups include, for example, thiophene,thianthrene, furan, pyran, isobenzofuran, chromene, xanthene,phenoxathiin, pyrrole, imidazole, pyrazole, isothiazole, isoxazole,pyridine, pyrazine, pyrimidine, pyridazine, indolizine, isoindole,indole, indazole, purine, quinolizine, isoquinoline, quinoline,plithalazine, naphthyridine, quinoxaline, quinazoline, cinnoline,pteridine, carbazole, carboline, phenanthridine, acridine, pyrimidine,phenanthroline, phenazine, phenarsazine, phenothiazine, furazan,phenoxazine, pyrrolidine, oxolane, thioiane, oxazole, piperidine,piperazine, morpholine, lactones, lactams such as azetidinones andpyrrolidinones, sultams, sultones, and the like. The heterocyclic ringcan be substituted at one or more positions with such substituents asdescribed above, as for example, halogen, alkyl, aralkyl, alkenyl,alkynyl, cycloalkyl, hydroxyl, amino, nitro, sulfhydryl, imino, amido,phosphate, phosphonate, phosphinate, carbonyl, carboxyl, silyl, ether,alkylthio, sulfonyl, ketone, aldehyde, ester, a heterocyclyl, anaromatic or heteroaromatic moiety, —CF₃, —CN, or the like.

As used herein, the term “nitro” means —NO₂; the term “halogen”designates —F, —Cl, —Br or —I; the term “sulfhydryl” means —SH; the term“hydroxyl” means —OH; and the term “sulfonyl” means —SO₂—.

A “phosphonamidite” can be represented in the general formula:

wherein R₉ and R₁₀ are as defined above, Q₂ represents O, S or N, andR₄₈ represents a lower alkyl or an aryl, Q₂ represents O, S or N.

A “phosphoramidite” can be represented in the general formula:

wherein R₉ and R₁₀ are as defined above, and Q₂ represents O, S or N.

A “phosphoryl” can in general be represented by the formula:

wherein Q₁ represented S or O, and R₄₆ represents hydrogen, a loweralkyl or an aryl. When used to substitute, for example, an alkyl, thephosphoryl group of the phosphorylalkyl can be represented by thegeneral formula:

wherein Q₁ represented S or O, and each R₄₆ independently representshydrogen, a lower alkyl or an aryl, Q₂ represents O, S or N. When Q₁ isan S, the phosphoryl moiety is a “phosphorothioate”.

The terms “polycyclyl” or “polycyclic group” refer to two or more rings(e.g., cycloalkyls, cycloalkenyls, cycloalkynyls, aryls and/orheterocyclyls) in which two or more carbons are common to two adjoiningrings, e.g., the rings are “fused rings”. Rings that are joined throughnon-adjacent atoms are termed “bridged” rings. Each of the rings of thepolycycle can be substituted with such substituents as described above,as for example, halogen, alkyl, aralkyl, alkenyl, alkynyl, cycloalkyl,hydroxyl, amino, nitro, sulthydryl, imino, amido, phosphate,phosphonate, phosphinate, carbonyl, carboxyl, silyl, ether, alkylthio,sulfonyl, ketone, aldehyde, ester, a heterocyclyl, an aromatic orheteroaromatic moiety, —CF₃, —CN, or the like.

The phrase “protecting group” as used herein means temporarysubstituents which protect a potentially reactive functional group fromundesired chemical transformations. Examples of such protecting groupsinclude esters of carboxylic acids, silyl ethers of alcohols, andacetals and ketals of aldehydes and ketones, respectively. The field ofprotecting group chemistry has been reviewed (Greene, T. W.; Wuts, P. G.M. Protective Groups in Organic Synthesis, 2^(nd) ed.; Wiley: New York,1991).

A “selenoalkly” refers to an alkyl group having a substituted selenogroup attached thereto. Exemplary “selenoethers” which may besubstituted on the alkyl are selected from one of —Se-alkyl,—Se-alkenyl, —Se-alkynyl, and —Se—(CH₂)_(m)—R₈, m and R₈ being definedabove.

As used herein, the term “substituted” is contemplated to include allpermissible substituents of organic compounds. In a broad aspect, thepermissible substituents include acyclic and cyclic, branched andunbranched, carbocyclic and heterocyclic, aromatic and nonaromaticsubstituents of organic compounds. Illustrative substituents include,for example, those described herein above. The permissible substituentscan be one or more and the same or different for appropriate organiccompounds. For purposes of this invention, the heteroatoms such asnitrogen may have hydrogen substituents and/or any permissiblesubstituents of organic compounds described herein which satisfy thevalences of the heteroatoms. This invention is not intended to belimited in any manner by the permissible substituents of organiccompounds.

It will be understood that “substitution” or “substituted with” includesthe implicit proviso that such substitution is in accordance withpermitted valence of the substituted atom and the substituent, and thatthe substitution results in a stable compound, e.g., which does notspontaneously undergo transformation such as by rearrangement,cyclization, elimination, etc.

The term “sulfamoyl” is art-recognized and includes a moiety that can berepresented by the general formula:

in which R₉ and R₁₀ are as defined above.

The term “sulfate” is art recognized and includes a moiety that can berepresented by the general formula:

in which R41 is as defined above.

The term “sulfonamido” is art recognized and includes a moiety that canbe represented by the general formula:

in which R₉ and R′₁₁ are as defined above.

The term “sulfonate” is art-recognized and includes a moiety that can berepresented by the general formula:

in which R41 is an electron pair, hydrogen, alkyl, cycloalkyl, or aryl.

The terms “sulfoxido” or “sulfinyl”, as used herein, refers to a moietythat can be represented by the general formula:

in which R₄₄ is selected from the group consisting of hydrogen, alkyl,alkenyl, alkynyl, cycloalkyl, heterocyclyl, aralkyl, or aryl.

Analogous substitutions can be made to alkenyl and alkynyl groups toproduce, for example, aminoalkenyls, arninoalkynyls, amidoalkenyls,amidoalkynyls, iminoalkenyls, iminoalkynyls, thioalkenyls, thioalkynyls,carbonyl-substituted alkenyls or alkynyls.

As used herein, the definition of each expression, e.g., alkyl, m, n,etc., when it occurs more than in any structure, is intended to beindepentent of its definition elsewhere in the same structure.

The terms triflyl, tosyl, mesyl, and nonaflyl are art-recognized andrefer to trifluoromethanesulfonyl, p-toluenesulfonyl, methanesufonyl,and nonafluorobutanesulfonyl groups, respectively. The terms triflate,tosylate, mesylate, and nonaflate are art-recognized and refer totrifluoromethanesulfonate ester, p-toluenesulfonate ester,methanesulfonate ester, and nonafluorobutanesulfonate ester functionalgroups and molecules that contain said groups, respectively.

The abbreviations Me, Et, Ph, Tf, Nf, Ts, Ms represent methyl, ethyl,phenyl, trifluoromethanesulfonyl, . nonafluorobutanesulfonyl,p-toluenesulfonyl and methanesulfonyl, respectively. A morecomprehensive list of the abbreviations utilized by organic chemists ofordinary skill in the art appears in the first issue of each volume ofthe Journal of Organic Chemistry; this list is typically presented in atable entitled Standard List of Abbreviations. The abbreviationscontained in said list, and all abbreviations utilized by organicchemists of ordinary skill in the art are hereby incorporated byreference.

Certain compounds of the present invention may exist in particulargeometric or stereoisomeric forms. The present invention contemplatesall such compounds, including cis- and trans-isomers, R- andS-enantiomers, diastereomers, (D)-isomers, (L)-isomers, the racemicmixtures thereof, and other mixtures thereof, as falling within thescope of the invention. Additional asymmetric carbon atoms may bepresent in a substituent such as an alkyl group. All such isomers, aswell as mixtures thereof, are intended to be included in this invention.

If, for instance, a particular enantiomer of a compound of the presentinvention is desired, it may be prepared by asymmetric synthesis, or byderivation with a chiral auxiliary, where the resulting diastereomericmixture is separated and the auxiliary group cleaved to provide the puredesired enantiomers. Alternatively, where the molecule contains a basicfunctional group, such as amino, or an acidic functional group, such ascarboxyl, diastereomeric salts may be formed with an appropriateoptically active acid or base, followed by resolution of thediastereomers thus formed by fractionsl crystallization orchromatographic means well known in the art, and subsequent recovery ofthe pure enantiomers.

Contemplated equivalents of the compounds described above includecompounds which otherwise correspond thereto, and which have the samegeneral properties thereof (e.g., the ability to inhibit hedgehogsignaling), wherein one or more simple variations of substituents aremade which do not adversely affect the efficacy of the compound. Ingeneral, the compounds of the present invention may be prepared by themethods illustrated in the general reaction schemes as, for example,described below, or by modifications thereof, using readily availablestarting materials, reagents and conventional synthesis procedures. Inthese reactions, it is also possible to make use of variants which arein themselves known, but are not mentioned here.

For purposes of this invention, the chemical elements are identified inaccordance with the Periodic Table of the Elements, CAS version,Handbook of Chemistry and Physics, 67th Ed., 1986-87, inside cover. Alsofor purposes of this invention, the term “hydrocarbon” is contemplatedto include all permissible compounds having at least one hydrogen andone carbon atom. In a broad aspect, the permissible hydrocarbons includeacyclic and cyclic, branched and unbranched, carbocyclic andheterocyclic, aromatic and nonaromatic organic compounds which can besubstituted or unsubstituted.

III. Exemplary Compounds of the Invention.

As described in further detail below, it is contemplated that thesubject methods can be carried out using a variety of different smallmolecules which can be readily identified, for example, by such drugscreening assays as described herein. For example, compounds useful inthe subject methods include compounds may be represented by generalformula (I):

wherein, as valence and stability permit,

-   Ar represents a substituted or unsubstituted aryl or heteroaryl    ring, preferably a substituted or unsubstituted phenyl ring;-   X represents, independently for each occurrence, N or CR′,    preferably N;-   X₁ represents, independently for each occurrence, N or CR″,    preferably N;-   R′ represents, independently for each occurrence, H, halogen, alkyl,    alkenyl, alkynyl, aryl, hydroxyl, alkoxyl, silyloxy, amino,    alkylamino, nitro, cyano, thiol, amino, imino, amido, phosphoryl,    phosphonate, carboxyl, carboxamide, anhydride, silyl, thioether,    alkylsulfonyl, arylsulfonyl, selenoether, acyl, aldehyde, ester,    carbamate, or carbonate, preferably H, halogen, alkyl, hydroxyl,    alkoxyl, or amino;-   R″ represents, independently for each occurrence, H, halogen, alkyl,    alkenyl, alkynyl, aryl, hydroxyl, alkoxyl, silyloxy, amino,    alkylamino, nitro, cyano, thiol, amino, imino, amido, phosphoryl,    phosphonate, carboxyl, carboxamide, anhydride, silyl, thioether,    alkylsulfonyl, arylsulfonyl, selenoether, acyl, aldehyde, ester,    carbamate, or carbonate, preferably H, halogen, alkyl, hydroxyl,    alkoxyl, or amino;-   R₁ represents a substituted or unsubstituted alkyl, alkenyl,    alkynyl, cycloalkyl, cycloalkylalkyl, heterocyclyl, heterocyclyl    alkyl, aryl, aralkyl, heteroaryl, or heteroaralkyl, preferably    alkyl, heteroaralkyl, or aralkyl (such as substituted or    unsubstituted benzyl or phenethyl);-   R₈ represents from 0-4 substituents on the ring to which it is    attached, independently selected from halogen, alkyl, alkenyl,    alkynyl, aryl, hydroxyl, ═O, ═S, alkoxyl, silyloxy, amino, nitro,    cyano, thiol, amino, alkylamino, imino, amido, phosphoryl,    phosphonate, carboxyl, carboxamide, anhydride, silyl, thioether,    alkylsulfonyl, arylsulfonyl, selenoether, acyl, aldehyde, ester,    carbamate, and carbonate, preferably lower alkyl or 0 substituents;    and-   R₉ represents from 0-4 substituents on the ring to which it is    attached, independently selected from halogen, alkyl alkenyl,    alkynyl, aryl, hydroxyl, alkoxyl, silyloxy, amino, nitro, cyano,    thiol, amino, alkylamino, imino, amido, phosphoryl, phosphonate,    carboxyl, carboxamide, anhydride, silyl, thioether, alkylsulfonyl,    arylsulfonyl, selenoether, acyl, aldehyde, ester, carbarnate, and    carbonate, preferably halogen, nitro, cyano, or lower alkyl, or 0    substituents,    or a pharmaceutically acceptable salt thereof.

Exemplary compounds of Formula I are shown in Scheme 1 below. In certainembodiments, compounds of Formula I of the invention do not include theo-chlorobenzyl substituted compound of Scheme 1.

In still other embodiments, the invention provides antagonists having astructure of Formula II:

wherein, as valence and stability permit,

-   A represents a pyridyl or dihydropyridyl ring;-   Ar represents a substituted or unsubstituted aryl or heteroaryl    ring, preferably a substituted or unsubstituted phenyl ring; and-   R₉ represents from 0-4 substituents on the ring to which it is    attached, independently selected from halogen, alkyl, alkenyl,    alkynyl, aryl, hydroxyl, alkoxyl, silyloxy, amino, nitro, cyano,    thiol, amino, alkylamino, imino, amido, phosphoryl, phosphonate,    carboxyl, carboxamide, anhydride, silyl, thioether, alkylsulfonyl,    arylsulfonyl, selenoether, acyl, aldehyde, ester, carbamate, and    carbonate, preferably H, halogen, alkyl, hydroxyl, alkoxyl,    thioether, acyl, alkylamino, or amino,    or a pharmaceutically acceptable salt thereof.

Exemplary compounds of Formula II are shown in Scheme 3 below. Incertain embodiments, compounds of Formula II of the invention do notinclude the methoxyphenyl-substituted compound at the top center ofScheme 3.

In yet other embodiments, the invention provides compounds having astructure of Formula III:

wherein, independently for each occurrence,

-   A represents a pyridyl or dihydropyridyl ring;-   Ar represents a substituted or unsubstituted aryl or heteroaryl    ring, preferably a substituted or unsubstituted phenyl ring; and-   R₂ represents a thioether, preferably substituted with an amido,    ester, carboxyl, or acyl, such as substituted or unsubstituted    benzoyl or aminocarbonyl;-   R₃ represents H, alkyl, alkenyl, alkynyl, or cycloalkyl; and-   R₄ represents a substituent selected from H, halogen, alkyl,    alkenyl, alkynyl, aryl, hydroxyl, alkoxyl, silyloxy, amino, nitro,    cyano, thiol, amino, alkylamino, imino, amido, phosphoryl,    phosphonate, carboxyl, carboxamide, anhydride, silyl, thioether,    alkylsulfonyl, arylsulfonyl, selenoether, acyl, aldehyde, ester,    carbamate, and carbonate, preferably H, halogen, alkyl, alkenyl,    alkynyl, hydroxyl, nitro, cyano, or acyl (e.g., one occurrence of    cyano and one occurrence of acyl, such as acetyl or other    alkylcarbonyl),    or a pharmaceutically acceptable salt thereof.

Exemplary compounds of Formula III are shown in Scheme 3 below. Incertain embodiments, compounds of Formula III of the invention do notinclude the methoxyphenyl-substituted compound at the top center ofScheme 3. In preferred compounds of Formulae II and III, when Arepresents a dihydropyridyl ring, the nitrogen atom and the Ar-bearingcarbon atom bear hydrogen substituents. In certain such embodiments, thedihydropyridyl ring bears an acyl, ester, carboxyl, carboxamide, orother carbonyl substituent, e.g., adjacent (vicinal) to the Arsubstituent.

In additional embodiments, the present invention provides compoundshaving a structure of Formula IV:

wherein, as valence and stability permit,

-   Ar represents a substituted or unsubstituted aryl or heteroaryl    ring, preferably a substituted or unsubstituted phenyl ring;-   W represents C═O, C═S, or SO₂;-   Har represents a substituted or unsubstituted heteroaryl ring,    preferably a substituted or unsubstituted pyridyl ring, such as    2-pyridyl, preferably substituted with a C₁-C₁₀ alkyl, such as an    unbranched alkyl at the 5-position of the pyridine ring;-   Ht represents OR or NR′″₂, such as a mono- or di-substituted amino    group, e.g., a dialkylamino or a cyclic amino group such as a    piperidyl, piperazinyl, or morpholino group;-   R represents H or lower alkyl; and-   R′″ represents H, lower alkyl, cycloalkyl, cycloalkylalkyl,    heterocyclyl, heterocyclyl alkyl, aryl, aralkyl, heteroaryl, or    heteroaralkyl, preferably aryl or aralkyl,    or a pharmaceutically acceptable salt thereof.

Exemplary compounds of Formula IV are shown in Scheme 4 below. Incertain embodiments, compounds of Formula IV of the invention do notinclude the pentylpyridine-piperidinamide compound (260 nM) of Scheme 4.

In still another embodiment, the present invention provides compoundshaving a structure of Formula V:

wherein, as valence and stability permit,

-   Ar represents a substituted or unsubstituted aryl or heteroaryl    ring, preferably a substituted or unsubstituted phenyl ring, such as    an ortho-hydroxyphenyl group optionally substituted with one or more    halogen, hydroxy, alkoxy, amino, alkylamino, alkyl, thiol,    alkylthio, ester, amido, carboxyl, nitro, or cyano substituents;-   Y represents OH or NR₂, preferably NH₂;-   Z represents O or S;-   R represents H or lower alkyl; and-   R₅ represents substituted or unsubstituted lower alkyl, preferably a    trihalomethyl group,    or a pharmaceutically acceptable salt thereof.

Exemplary compounds of Formula V are shown in Scheme 5 below.

In still yet another embodiment, the invention provides compounds havinga structure of Formula VI:

wherein, as valence and stability permit,

-   Ar, independently for each occurrence, represents a substituted or    unsubstituted aryl or heteroaryl ring, preferably a substituted or    unsubstituted phenyl ring;-   W represents C═O, C═S, or SO₂;-   X represents, independently for each occurrence, N or CR′,    preferably N;-   X₁ represents, independently for each occurrence, N or CR″,    preferably CR;-   R represents H or lower alkyl; and-   R′ represents, independently for each occurrence, H, halogen, alkyl,    alkenyl, alkynyl, aryl, hydroxyl, alkoxyl, silyloxy, amino,    alkylamino, nitro, cyano, thiol, amino, imino, amido, phosphoryl,    phosphonate, carboxyl, carboxamide, anhydride, silyl, thioether,    alkylsulfonyl, arylsulfonyl, selenoether, acyl, aldehyde, ester,    carbamate, or carbonate, preferably H, halogen, alkyl, hydroxyl,    alkoxyl, or amino;-   R″ represents, independently for each occurrence, H, halogen, alkyl,    alkenyl, alkynyl, aryl, hydroxyl, alkoxyl, silyloxy, amino,    alkylamino, nitro, cyano, thiol, amino, imino, amido, phosphoryl,    phosphonate, carboxyl, carboxamide, anhydride, silyl, thioether,    alkylsulfonyl, arylsulfonyl, selenoether, acyl, aldehyde, ester,    carbamate, or carbonate, preferably H, halogen, alkyl, hydroxyl,    alkoxyl, or amino; and-   R₉ represents from 0-4 substituents on the ring to which it is    attached, independently selected from halogen, alkyl, alkenyl,    alkynyl, aryl, hydroxyl, alkoxyl, silyloxy, amino, nitro, cyano,    thiol, amino, alkylamino, imino, amido, phosphoryl, phosphonate,    carboxyl, carboxamide, anhydride, silyl, thioether, alkylsulfonyl,    arylsulfonyl, selenoether, acyl, aldehyde, este, carbamate, and    carbonate, preferably from halogen, alkyl, cyano, and nitro,    or a pharmaceutically acceptable salt thereof.

Exemplary compounds of Formula VI are shown in Scheme 6 below.

Because compounds having structures of Formulae V and VI appear to actdownstream of smoothened, these compounds can be used to inhibit thehedgehog pathway in cells that have a smoothened gain-of-functionmutation that renders them at least partially resistant to inhibitorsthat act directly upon hedgehog, patched, or smoothened. Accordingly,the present invention contemplates hedgehog antagonists that inhibit thehedgehog pathway in a smoothened gain-of-function cell with an IC₅₀within a factor of 5, or even a factor of 2 or 1, of the IC₅₀ with whichthey inhibit the hedgehog pathway in a patched-null cell. Suchantagonists are suitable not only for normal cells, or cells withaberrant activity or expression of hedgehog, patched, or smoothened, butalso for cells with defects in the hedgehog pathway downstream of theseproteins.

In certain embodiments, an antagonist has the structure of Formula VII:

wherein, as valence and stability permit,

X and Z, independently, represent —N(R₇)—, —O—, —S—, —(R₇)N—N(R₇)—,—ON(R₇)—, or a direct bond, preferably —N(R₇)—, —O—, —S—, or a directbond;

Y represents —C(═O)—, —C(═S)—, —C(═NR₇)—, SO₂, or SO, preferably—C(═O)—, SO₂, or —C(═S)—;

A represents O, S, or NR7, preferably O or NH, and most preferably NH;

G represents a cycloalkyl, heterocyclyl, aryl, or heteroaryl ring fusedto the ring to which it is attached, preferably an aryl or heteroarylring.

Ar represents a substituted or unsubstituted aryl or heteroaryl ring,such as a substituted or unsubstituted phenyl ring;

R₁ represents H or substituted or unsubstituted alkyl, alkenyl, alkynyl,aryl, heteroaryl, heterocyclyl, or cycloalkyl, including polycyclicgroups;

R₂ represents from 0-4 substituents on the ring to which it is attached,such as halogen, lower alkyl, lower alkenyl, aryl, heteroaryl, carbonylgroup (e.g., ester, carboxyl, or formyl), thiocarbonyl (e.g., thioester,thiocarboxylate, or thioformate), ketone, aldehyde, amino, acylamino,amido, amidino, cyano, nitro, azido, sulfonyl, sulfoxido, sulfate,sulfonate, sulfamoyl, sulfonamido, phosphoryl, phosphonate, phosphinate,J-R₈, J-OH, J-lower alkyl, J-lower alkenyl, J-R₈, J-SH, J-NH₂, protectedforms of the above, or any two R₂, when occurring more than once in acyclic or polycyclic structure, can be taken together form a 4- to8-membered cycloalkyl, aryl, or heteroaryl;

R₇, independently for each occurrence, represents H, lower alkyl (e.g.,substituted or unsubstituted), J-cycloalkyl (e.g., substituted orunsubstituted), J-heterocyclyl (e.g., substituted or unsubstituted),J-aryl (e.g., substituted or unsubstituted), J-heteroaryl (e.g.,substituted or unsubstituted);

R₈, independently for each occurrence, represents H, lower alkyl (e.g.,substituted or unsubstituted), cycloalkyl (e.g., substituted orunsubstituted), heterocyclyl (e.g., substituted or unsubstituted), aryl(e.g., substituted or unsubstituted), or heteroaryl (e.g., substitutedor unsubstituted); and

J represents, independently for each occurrence, a chain having from 0-8(preferably from 0-4) units selected from CK₂, NK, O, and S, wherein Krepresents, independently for each occurrence, H or lower alkyl.

In certain embodiments, at least one of Z and X is not a direct bond. Incertain embodiments X-Y-Z includes an amide, urea, or sulfonamide. Incertain embodiments, X is selected from —N(R₈)—, —O—, —S—, andpreferably represents NH.

In certain embodiments, R₁ includes an aryl or heteroaryJ ring,optionally substituted with from 1-5 substituents, such as nitro,halogen, cyano, lower alkyl, acylamino (e.g., R₈—C(═O)NH—), alkoxy,alkylamino, a substituted or unsubstituted cycloalkyl, heterocyclyl,aryl, or heteroaryl fused to the aryl or heteroaryl ring.

In certain embodiments, X and the ring comprising A are disposed on Arin a meta (i.e., 1,3) relationship.

In certain embodiments, G represents a phenyl or piperidine ring.

In certain embodiments, J is absent.

In certain embodiments, R₂ represents from 1-4 substituents selectedfrom halogen, cyano, nitro, alkoxy, amino, acylamino (e.g.,R₈—C(═O)NH—), a substituted or unsubstituted cycloalkyl, heterocyclyl,aryl, or heteroaryl fused to G, and substituted or unsubstituted loweralkyl.

In certain embodiments, the subject compound is selected from thecompounds depicted in FIG. 32.

In certain embodiments, compounds useful in the present invention may berepresented by Formula VIIa:

wherein, as valence and stability permit,

X and Z, independently, represent —N(R₇)—, —O—, —S—, —(R₇)N—N(R₇)—,—ON(R₇)—, or a direct bond, preferably —N(R₇)—, —O—, —S—, or a directbond;

Y represents —C(═O)—, —C(═S)—, —C(═NR₇)—, SO₂, or SO, preferably—C(═O)—, SO₂, or —C(═S)—;

A represents O, S, or NR₇, preferably O or NH, and most preferably NH;

G represents a cycloalkyl, heterocyclyl, aryl, or heteroaryl ring fusedto the ring to which it is attached, preferably an aryl or heteroarylring.

R₁ represents H or substituted or unsubstituted alkyl, alkenyl, alkynyl,aryl, heteroaryl, heterocyclyl, or cycloalkyl, including polycyclicgroups;

R₂ represents from 0-4 substituents on the ring to which it is attached,such as halogen, lower alkyl, lower alkenyl, aryl, heteroaryl, carbonylgroup (e.g., ester, carboxyl, or formyl), thiocarbonyl (e.g., thioester,thiocarboxylate, or thioformate), ketone, aldehyde, amino, acylamino,amido, amidino, cyano, nitro, azido, sulfonyl, sulfoxido, sulfate,sulfonate, sulfamoyl, sulfonamido, phosphoryl, phosphonate, phosphinate,J-R₈, J-OH, J-lower alkyl, J-lower alkenyl, J-R₈, J-SH, J-NH₂, protectedforms of the above, or any two R₂, when occurring more than once in acyclic or polycyclic structure, can be taken together form a 4- to8-membered cycloalkyl, aryl, or heteroaryl;

R₃ represents from 0-4 substituents on the ring to which it is attached,such as halogen, hydroxyl, alkoxy, amino, alkylamino, cyano, nitro,substituted or unsubsituted lower alkyl, and acyl, preferably halogen,lower alkoxy, or substituted or unsubstituted lower alkyl;

R₇, independently for each occurrence, represents H, lower alkyl (e.g.,substituted or unsubstituted), J-cycloalkyl (e.g., substituted orunsubstituted), J-heterocyclyl (e.g., substituted or unsubstituted),J-aryl (e.g., substituted or unsubstituted), J-heteroaryl (e.g.,substituted or unsubstituted);

R₈, independently for each occurrence, represents H, lower alkyl (e.g.,substituted or unsubstituted), cycloalkyl (e.g., substituted orunsubstituted), heterocyclyl (e.g., substituted or unsubstituted), aryl(e.g., substituted or unsubstituted), or heteroaryl (e.g., substitutedor unsubstituted); and

J represents, independently for each occurrence, a chain having from 0-8(preferably from 0-4) units selected from CK₂, NK, 0, and S, wherein Krepresents, independently for each occurrence, H or lower alkyl.

In certain embodiments, at least one of Z and X is not a direct bond. Incertain embodiments, X-Y-Z includes an amide, urea, or sulfonamide. Incertain embodiments, X is selected from —N(R₈)—, —O—, —S—, andpreferably represents NH.

In certain embodiments, R₁ includes an aryl or heteroaryl ring,optionally substituted with from 1-5 substituents, such as nitro,halogen, cyano, lower alkyl, acylamino (e.g., R₈—C(═O)NH—), alkoxy,alkylamino, a substituted or unsubstituted cycloalkyl, heterocyclyl,aryl, or heteroaryl fused to the aryl or heteroaryl ring.

In certain embodiments, G represents a phenyl or piperidine ring.

In certain embodiments, J is absent.

In certain embodiments, R₂ represents from 1-4 substituents selectedfrom halogen, cyano, nitro, alkoxy, amino, acylamino (e.g.,R₈—C(═O)NH—), a substituted or unsubstituted cycloalkyl, heterocyclyl,aryl, or heteroaryl fused to G, and substituted or unsubstituted loweralkyl.

In certain embodiments, R₃ includes a substituent, such as a substitutedor unsubstituted alkyl or a halogen, at a position para either to X orto the ring including A.

In certain embodiments, the subject compound is selected from thecompounds depicted in FIG. 32.

As described in further detail below, it is contemplated that thesubject methods can be carried out using a variety of different smallmolecules which can be readily identified, for example, by such drugscreening assays as described herein. For example, compounds useful inthe subject methods include compounds may be represented by FormulaVIIb:

wherein, as valence and stability permit,

X and Z, independently, represent —N(R₇)—, —O—, —S—, —(R₇)N—N(R₇)—,—ON(R₇)—, or a direct bond, preferably —N(R₇)—, —O—, —S—, or a directbond;

Y represents —C(═O)—, —C(═S)—, —C(═NR₇)—, SO₂, or SO, preferably—C(═O)—, SO₂, or —C(═S)—;

A represents O, S, or NR₇, preferably O or NH, and most preferably NH;

G represents a cycloalkyl, heterocyclyl, aryl, or heteroaryl ring fusedto the ring to which it is attached, preferably an aryl or heteroarylring;

Q is absent, or represents CK₂, NK, O, and S, wherein K represents,independently for each occurrence, H or lower alkyl;

Ar represents a substituted or unsubstituted aryl or heteroaryl ring,such as a substituted or unsubstituted phenyl ring;

R₁ represents H or substituted or unsubstituted alkyl, alkenyl, alkynyl,aryl, heteroaryl, heterocyclyl, or cycloalkyl, including polycyclicgroups;

R₂ represents from 0-4 substituents on the ring to which it is attached,such as halogen, lower alkyl, lower alkenyl, aryl, heteroaryl, carbonylgroup (e.g., ester, carboxyl, or formyl), thiocarbonyl (e.g., thioester,thiocarboxylate, or thioformate), ketone, aldehyde, amino, acylamino,amido, amidino, cyano, nitro, azido, sulfonyl, sulfoxido, sulfate,sulfonate, sulfamoyl, sulfonamido, phosphoryl, phosphonate, phosphinate,J-R₈, J-OH, J-lower alkyl, J-lower alkenyl, J-R₈, J-SH, J-NH₂, protectedforms of the above, or any two R₂, when occurring more than once in acyclic or polycyclic structure, can be taken together form a 4- to8-membered cycloalkyl, aryl, or heteroaryl;

R₇, independently for each occurrence, represents H, lower alkyl (e.g.substituted or unsubstituted), J-cycloalkyl (e.g., substituted orunsubstituted), J-heterocyclyl (e.g., substituted or unsubstituted),J-aryl (e.g., substituted or unsubstituted), J-heteroaryl (e.g.,substituted or unsubstituted);

R₈, independently for each occurrence, represents H, lower alkyl (e.g.,substituted or unsubstituted), cycloalkyl (e.g., substituted orunsubstituted), heterocyclyl (e.g., substituted or unsubstituted), aryl(e.g., substituted or unsubstituted), or heteroaryl (e.g., substitutedor unsubstituted); and

J represents, independently for each occurrence, a chain having from 0-8(preferably from 0-4) units selected from CK₂, NK, O, and S.

In certain embodiments, at least one of Z and X is not a direct bond. Incertain embodiments, X-Y-Z includes an amide, urea, or sulfonamide. Incertain embodiments, X is selected from —N(R₈)—, —O—, —S—, andpreferably represents NH.

In certain embodiments, R₁ includes an aryl or heteroaryl ring,optionally substituted with from 1-5 substituents, such as nitro,halogen, cyano, lower alkyl, acylamino (e.g., R₈—C(═O)NH—), alkoxy,alkylamino, a substituted or unsubstituted cycloalkyl, heterocyclyl,aryl, or heteroaryl fuised to the aryl or heteroaryl ring.

In certain embodiments, X and the ring comprising A are disposed on Arin a meta (i.e., 1,3) relationship.

In certain embodiments, G represents a phenyl or piperidine ring.

In certain embodiments, J is absent.

In certain embodiments, R₂ represents from 1-4 substituents selectedfrom halogen, cyano, nitro, alkoxy, amino, acylamino (e.g.,R₈—C(═O)NH—), a substituted or unsubstituted cycloalkyl, heterocyclyl,aryl, or heteroaryl fused to G, and substituted or unsubstituted loweralkyl.

In certain embodiments, the subject compound is selected from thecompounds depicted in FIG. 32.

In certain embodiments, compounds useful in the present invention may berepresented by Formula VIIc:

wherein, as valence and stability permit,

X and Z, independently, represent —N(R₇)—, —O—, —S—, —(R₇)N—N(R₇)—,—ON(R₇)—, or a direct bond, preferably —N(R₇)—, —O—, —S—, or a directbond;

Y represents —C(═O)—, —C(═S)—, —C(═NR₇)—, SO₂, or SO, preferably—C(═O)—, SO₂, or —C(═S)—;

A represents O, S, or NR₇, preferably O or NH, and most preferably NH;

G represents a cycloalkyl, heterocyclyl, aryl, or heteroaryl ring fusedto the ring to which it is attached, preferably an aryl or heteroarylring.

Q is absent, or represents CK₂, NK, O, and S, wherein K represents,independently for each occurrence, H or lower alkyl;

R₁ represents H or substituted or unsubstituted alkyl, alkenyl, alkynyl,aryl, heteroaryl, heterocyclyl, or cycloalkyl, including polycyclicgroups;

R₂ represents from 0-4 substituents on the ring to which it is attached,such as halogen, lower alkyl, lower alkenyl, aryl, heteroaryl, carbonylgroup (e.g., ester, carboxyl, or formyl), thiocarbonyl (e.g., thioester,thiocarboxylate, or thioformate), ketone, aldehyde, amino, acylamino,amido, amidino, cyano, nitro, azido, sulfonyl, sulfoxido, sulfate,sulfonate, sulfamoyl, sulfonamido, phosphoryl, phosphonate, phosphinate,J-R₈, J-OH, J-lower alkyl, J-lower alkenyl, J-R₈, J-SH, J-NH₂, protectedforms of the above, or any two R₂, when occurring more than once in acyclic or polycyclic structure, can be taken together forrn a 4- to8-membered cycloalkyl, aryl, or heteroaryl;

R₃ represents from 0-4 substituents on the ring to which it is attached,such as halogen, hydroxyl, alkoxy, amino, alkylamino, cyano, nitro,substituted or unsubsituted lower alkyl, and acyl, preferably halogen,lower alkoxy, or substituted or unsubstituted lower alkyl;

R₇, independently for each occurrence, represents H, lower alkyl (e.g.,substituted or unsubstituted), J-cycloalkyl (e.g., substituted orunsubstituted), J-heterocyclyl (e.g., substituted or unsubstituted),J-aryl (e.g., substituted or unsubstituted), J-heteroaryl (e.g.,substituted or unsubstituted);

R₈, independently for each occurrence, represents H, lower alkyl (e.g.,substituted or unsubstituted), cycloalkyl (e.g., substituted orunsubstituted), heterocyclyl (e.g., substituted or unsubstituted), aryl(e.g., substituted or unsubstituted), or heteroaryl (e.g., substitutedor unsubstituted); and

J represents, independently for each occurrence, a chain having from 0-8(preferably from 0-4) units selected from CK₂, NK, O, and S.

In certain embodiments, at least one of Z and X is not a direct bond. Incertain embodiments, X-Y-Z includes an amide, urea, or sulfonamide. Incertain embodiments, X is selected from —N(R₈)—, —O—, —S—, andpreferably represents NH.

In certain embodiments, R₁ includes an aryl or heteroaryl ring,optionally substituted with from 1-5 substituents, such as nitro,halogen, cyano, lower alkyl, acylamino (e.g., R₈—C(═O)NH—), alkoxy,alkylamino, a substituted or unsubstituted cycloalkyl, heterocyclyl,aryl, or heteroaryl fused to the aryl or heteroaryl ring.

In certain embodiments, G represents a phenyl or piperidine ring.

In certain embodiments, J is absent.

In certain embodiments, R₂ represents from 1-4 substituents selectedfrom halogen, cyano, nitro, alkoxy, amino, acylamino (e.g.,R₈—C(═O)NH—), a substituted or unsubstituted cycloalkyl, heterocyclyl,aryl, or heteroaryl fused to G, and substituted or unsubstituted loweralkyl.

In certain embodiments, R₃ includes a substituent, such as a substitutedor unsubstituted alkyl or a halogen, at a position para either to X orto the ring including A.

Compounds of Formula VII include

In certain embodiments, the subject antagonists can be chosen on thebasis of their selectively for the hedgehog pathway. This selectivitycan be for the hedgehog pathway versus other pathways, or forselectivity between particular hedgehog pathways, e.g., ptc-1, ptc-2,etc.

In certain preferred embodiments, the subject inhibitors inhibit ptcloss-of-function, hedgehog gain-of-function, or smoothenedgain-of-function mediated signal transduction with an ED₅₀ of 1 mM orless, more preferably of 1 μM or less, and even more preferably of 1 nMor less. Similarly, in certain preferred embodiments, the subjectinhibitors inhibit activity of the hedgehog pathway with a K_(i) lessthan 10 nM, preferably less than 1 nM, even more preferably less than0.1 nM.

In particular embodiments, the small molecule is chosen for use becauseit is more selective for one patched isoform over the next, e.g.,10-fold, and more preferably at least 100- or even 1000-fold moreselective for one patched pathway (ptc-1, ptc-2) over another.

In certain embodiments, a compound which is an antagonist of thehedgehog pathway is chosen to selectively antagonize hedgehog activityover protein kinases other than PKA, such a PKC, e.g., the compoundmodulates the activity of the hedgehog pathway at least an order ofmagnitude more strongly than it modulates the activity of anotherprotein kinase, preferably at least two orders of magnitude morestrongly, even more preferably at least three orders of magnitude morestrongly. Thus, for example, a preferred inhibitor of the hedgehogpathway may inhibit hedgehog activity with a K_(i) at least an order ofmagnitude lower than its K_(i) for inhibition of PKC, preferably atleast two orders of magnitude lower, even more preferably at least threeorders of magnitude lower. In certain embodiments, the K_(i) for PKAinhibition is less than 10 nM, preferably less than 1 nM, even morepreferably less than 0.1 nM.

IV. Exemplary Applications of Method and Compositions

Another aspect of the present invention relates to a method ofmodulating a differentiated state, survival, and/or proliferation of acell having a ptc loss-of-function, hedgehog gain-of-function, orsmoothened gain-of-function, by contacting the cells with a hedgehogantagonist according to the subject method and as the circumstances maywarrant.

For instance, it is contemplated by the invention that, in light of thefindings of an apparently broad involvement of hedgehog, ptc, andsmoothened in the formation of ordered spatial arrangements ofdifferentiated tissues in vertebrates, the subject method could be usedas part of a process for generating and/or maintaining an array ofdifferent vertebrate tissue both in vitro and in vivo. The hedgehogantagonist, whether inductive or anti-inductive with respectproliferation or differentiation of a given tissue, can be, asappropriate, any of the preparations described above.

For example, the present method is applicable to cell culture techniqueswherein, whether for genetic or biochemical reasons, the cells have aptc loss-of-function, hedgehog gain-of-function, or smoothenedgain-of-function phenotype. In vitro neuronal culture systems haveproved to be fundamental and indispensable tools for the study of neuraldevelopment, as well as the identification of neurotrophic factors suchas nerve growth factor (NGF), ciliary trophic factors (CNTF), and brainderived neurotrophic factor (BDNF). One use of the present method may bein cultures of neuronal stem cells, such as in the use of such culturesfor the generation of new neurons and glia. In such embodiments of thesubject method, the cultured cells can be contacted with a hedgehogantagonist of the present invention in order to alter the rate ofproliferation of neuronal stem cells in the culture and/or alter therate of differentiation, or to maintain the integrity of a culture ofcertain terminally differentiated neuronal cells. In an exemplaryembodiment, the subject method can be used to culture, for example,sensory neurons or, alternatively, motomeurons. Such neuronal culturescan be used as convenient assay systems as well as sources ofimplantable cells for therapeutic treatments.

According to the present invention, large numbers of non-tumorigenicneural progenitor cells can be perpetuated in vitro and their rate ofproliferation and/or differentiation can be affected by contact withhedgehog antagonists of the present invention. Generally, a method isprovided comprising the steps of isolating neural progenitor cells froman animal, perpetuating these cells in vitro or in vivo, preferably inthe presence of growth factors, and regulating the differentiation ofthese cells into particular neural phenotypes, e.g., neurons and glia,by contacting the cells with a hedgehog antagonist.

Progenitor cells are thought to be under a tonic inhibitory influencethat maintains the progenitors in a suppressed state until theirdifferentiation is required. However, recent techniques have beenprovided which permit these cells to be proliferated, and unlike neuronsthat are terminally differentiated and therefore non-dividing, they canbe produced in unlimited number and are highly suitable fortransplantation into heterologous and autologous hosts withneurodegenerative diseases.

By “progenitor” it is meant an oligopotent or multipotent stem cell thatis able to divide without limit and, under specific conditions, canproduce daughter cells that terminally differentiate such as intoneurons and glia. These cells can be used for transplantation into aheterologous or autologous host. By heterologous is meant a host otherthan the animal from which the progenitor cells were originally derived.By autologous is meant the identical host from which the cells wereoriginally derived.

Cells can be obtained from embryonic, post-natal, juvenile or adultneural tissue from any animal. By any animal is meant any multicellularanimal that contains nervous tissue. More particularly, is meant anyfish, reptile, bird, amphibian or mammal and the like. The mostpreferable donors are mammals, especially mice and humans.

In the case of a heterologous donor animal, the animal may beeuthanized, and the brain and specific area of interest removed using asterile procedure. Brain areas of particular interest include any areafrom which progenitor cells can be obtained which will serve to restorefunction to a degenerated area of the host's brain. These regionsinclude areas of the central nervous system (CNS) including the cerebralcortex, cerebellum, midbrain, brainstem, spinal cord and ventriculartissue, and areas of the peripheral nervous system (PNS) including thecarotid body and the adrenal medulla. More particularly, these areasinclude regions in the basal ganglia, preferably the striatum whichconsists of the caudate and putamen, or various cell groups such as theglobus pallidus, the subthalamic nucleus, the nucleus basalis which isfound to be degenerated in Alzheimer's Disease patients, or thesubstantia nigra pars compacta which is found to be degenerated inParkinson's Disease patients.

Human heterologous neural progenitor cells may be derived from fetaltissue obtained from elective abortion, or from a post-natal, juvenileor adult organ donor. Autologous neural tissue can be obtained bybiopsy, or from patients undergoing neurosurgery in which neural tissueis removed, in particular during epilepsy surgery, and more particularlyduring temporal lobectomies and hippocampalectomies.

Cells can be obtained from donor tissue by dissociation of individualcells from the connecting extracellular matrix of the tissue.Dissociation can be obtained using any known procedures, includingtreatment with enzymes such as trypsin, collagenase and the like, or byusing physical methods of dissociation such as with a blunt instrumentor by mincing with a scalpel to a allow outgrowth of specific cell typesfrom a tissue. Dissociation of fetal cells can be carried out in tissueculture medium, while a preferable medium for dissociation of juvenileand adult cells is artificial cerebral spinal fluid (aCSF). Regular aCSFcontains 124 mM NaCl, 5 mM KCl, 1.3 mM MgCl₂, 2 mM CaCl₂, 26 mM NaHCO₃,and 10 mM D-glucose. Low Ca²⁺ aCSF contains the same ingredients exceptfor MgCl₂ at a concentration of 3.2 mM and CaCl₂ at a concentration of0.1 mM.

Dissociated cells can be placed into any known culture medium capable ofsupporting cell growth, including MEM, DMEM, RPMI, F-12, and the like,containing supplements which are required for cellular metabolism suchas glutamine and other amino acids, vitamins, minerals and usefulproteins such as transferrin and the like. Medium may also containantibiotics to prevent contamination with yeast, bacteria and fungi suchas penicillin, streptomycin, gentamicin and the like. In some cases, themedium may contain serum derived from bovine, equine, chicken and thelike. A particularly preferable medium for cells is a mixture of DMEMand F-12.

Conditions for culturing should be close to physiological conditions.The pH of the culture media should be close to physiological pH,preferably between pH 6-8, more preferably close to pH 7, even moreparticularly about pH 7.4. Cells should be cultured at a temperatureclose to physiological temperature, preferably between 30° C.-40° C.,more preferably between 32° C.-38° C., and most preferably between 35°C.-37° C.

Cells can be grown in suspension or on a fixed substrate, butproliferation of the progenitors is preferably done in suspension togenerate large numbers of cells by formation of “neurospheres” (see, forexample, Reynolds et al. (1992) Science 255:1070-1709; and PCTPublications WO93/01275, WO94/09119, WO94/10292, and WO94/16718). In thecase of propagating (or splitting) suspension cells, flasks are shakenwell and the neurospheres allowed to settle on the bottom corner of theflask. The spheres are then transferred to a 50 ml centrifuge tube andcentrifuged at low speed. The medium is aspirated, the cells resuspendedin a small amount of medium with growth factor, and the cellsmechanically dissociated and resuspended in separate aliquots of media.

Cell suspensions in culture medium are supplemented with any growthfactor which allows for the proliferation of progenitor cells and seededin any receptacle capable of sustaining cells, though as set out above,preferably in culture flasks or roller bottles. Cells typicallyproliferate within 3-4 days in a 37° C. incubator, and proliferation canbe reinitiated at any time after that by dissociation of the cells andresuspension in fresh medium containing growth factors.

In the absence of substrate, cells lift off the floor of the flask andcontinue to proliferate in suspension forming a hollow sphere ofundifferentiated cells. After approximately 3-10 days in vitro, theproliferating clusters (neurospheres) are fed every 2-7 days, and moreparticularly every 2-4 days by gentle centrifugation and resuspension inmedium containing growth factor.

After 6-7 days in vitro, individual cells in the neurospheres can beseparated by physical dissociation of the neurospheres with a bluntinstrument, more particularly by triturating the neurospheres with apipette. Single cells from the dissociated neurospheres are suspended inculture medium containing growth factors, and differentiation of thecells can be control in culture by plating (or resuspending) the cellsin the presence of a hedgehog antagonist.

To further illustrate other uses of the subject hedgehog antagonists, itis noted that intracerebral grafting has emerged as an additionalapproach to central nervous system therapies. For example, one approachto repairing damaged brain tissues involves the transplantation of cellsfrom fetal or neonatal animals into the adult brain (Dunnett et al.(1987) J Exp Biol 123:265-289; and Freund et al. (1985) J Neurosci5:603-616). Fetal neurons from a variety of brain regions can besuccessfully incorporated into the adult brain, and such grafts canalleviate behavioral defects. For example, movement disorder induced bylesions of dopaminergic projections to the basal ganglia can beprevented by grafts of embryonic dopaminergic neurons. Complex cognitivefunctions that are impaired after lesions of the neocortex can also bepartially restored by grafts of embryonic cortical cells. The subjectmethod can be used to regulate the growth state in the culture, or wherefetal tissue is used, especially neuronal stem cells, can be used toregulate the rate of differentiation of the stem cells.

Stem cells useful in the present invention are generally known. Forexample, several neural crest cells have been identified, some of whichare multipotent and likely represent uncommitted neural crest cells, andothers of which can generate only one type of cell, such as sensoryneurons, and likely represent committed progenitor cells. The role ofhedgehog antagonists employed in the present method to culture such stemcells can be to regulate differentiation of the uncommitted progenitor,or to regulate further restriction of the developmental fate of acommitted progenitor cell towards becoming a terminally differentiatedneuronal cell. For example, the present method can be used in vitro toregulate the differentiation of neural crest cells into glial cells,schwann cells, chromaffin cells, cholinergic sympathetic orparasympathetic neurons, as well as peptidergic and serotonergicneurons. The hedgehog antagonists can be used alone, or can be used incombination with other neurotrophic factors that act to moreparticularly enhance a particular differentiation fate of the neuronalprogenitor cell.

In addition to the implantation of cells cultured in the presence of thesubject hedgehog antagonists, yet another aspect of the presentinvention concerns the therapeutic application of a hedgehog antagonistto regulate the growth state of neurons and other neuronal cells in boththe central nervous system and the peripheral nervous system. Theability of ptc, hedgehog, and smoothened to regulate neuronaldifferentiation during development of the nervous system and alsopresumably in the adult state indicates that, in certain instances, thesubject hedgehog antagonists can be expected to facilitate control ofadult neurons with regard to maintenance, functional performance, andaging of normal cells; repair and regeneration processes in chemicallyor mechanically lesioned cells; and treatment of degeneration in certainpathological conditions. In light of this understanding, the presentinvention specifically contemplates applications of the subject methodto the treatment protocol of (prevention and/or reduction of theseverity of) neurological conditions deriving from: (i) acute, subacute,or chronic injury to the nervous system, including traumatic injury,chemical injury, vascular injury and deficits (such as the ischemiaresulting from stroke), together with infectious/inflammatory andtumor-induced injury; (ii) aging of the nervous system includingAlzheimer's disease; (iii) chronic neurodegenerative diseases of thenervous system, including Parkinson's disease, Huntington's chorea,amylotrophic lateral sclerosis and the like, as well as spinocerebellardegenerations; and (iv) chronic immunological diseases of the nervoussystem or affecting the nervous system, including multiple sclerosis.

As appropriate, the subject method can also be used in generating nerveprostheses for the repair of central and peripheral nerve damage. Inparticular, where a crushed or severed axon is intubulated by use of aprosthetic device, hedgehog antagonists can be added to the prostheticdevice to regulate the rate of growth and regeneration of the dendridicprocesses. Exemplary nerve guidance channels are described in U.S. Pat.Nos. 5,092,871 and 4,955,892.

In another embodiment, the subject method can be used in the treatmentof neoplastic or hyperplastic transformations such as may occur in thecentral nervous system. For instance, the hedgehog antagonists can beutilized to cause such transformed cells to become either post-mitoticor apoptotic. The present method may, therefore, be used as part of atreatment for, e.g., malignant gliomas, meningiomas, medulloblastomas,neuroectodermal tumors, and ependymomas.

In a preferred embodiment, the subject method can be used as part of atreatment regimen for malignant medulloblastoma and other primary CNSmalignant neuroectodermal tumors.

In certain embodiments, the subject method is used as part of treatmentprogram for medulloblastoma. Medulloblastoma, a primary brain tumor, isthe most common brain tumor in children. A medulloblastoma is aprimitive neuroectodermal tumor arising in the posterior fossa. Theyaccount for approximately 25% of all pediatric brain tumors (Miller).Histologically, they are small round cell tumors commonly arranged intrue rosettes, but may display some differentiation to astrocytes,ependymal cells or neurons (Rorke; Kleihues). PNET's may arise in otherareas of the brain including the pineal gland (pineoblastoma) andcerebrum. Those arising in the supratentorial region generally fareworse than their PF counterparts.

Medulloblastoma/PNET's are known to recur anywhere in the CNS afterresection, and can even metastasize to bone. Pretreatment evaluationshould therefore include an examination of the spinal cord to excludethe possibility of “dropped metastases”. Gadolinium-enhanced MRI haslargely replaced myelography for this purpose, and CSF cytology isobtained postoperatively as a routine procedure.

In other embodiments, the subject method is used as part of treatmentprogram for ependymomas. Ependymomas account for approximately 10% ofthe pediatric brain tumors in children. Grossly, they are tumors thatarise from the ependymal lining of the ventricles and microscopicallyform rosettes, canals, and perivascular rosettes. In the CHOP series of51 children reported with ependymomas, ¾ were histologically benign.Approximately ⅔ arose from the region of the 4th ventricle. One thirdpresented in the supratentorial region. Age at presentation peaksbetween birth and 4 years, as demonstrated by SEER data as well as datafrom CHOP. The median age is about 5 years. Because so many childrenwith this disease are babies, they often require multimodal therapy.

Yet another aspect of the present invention concerns the observation inthe art that ptc, hedgehog, and/or smoothened are involved inmorphogenic signals involved in other vertebrate organogenic pathways inaddition to neuronal differentiation as described above, having apparentroles in other endodermal patterning, as well as both mesodermal andendodennal differentiation processes. Thus, it is contemplated by theinvention that compositions comprising hedgehog antagonists can also beutilized for both cell culture and therapeutic methods involvinggeneration and maintenance of non-neuronal tissue.

In one embodiment, the present invention makes use of the discovery thatptc, hedgehog, and smoothened are apparently involved in controlling thedevelopment of stem cells responsible for formation of the digestivetract, liver, lungs, and other organs which derive from the primitivegut. Shh serves as an. inductive signal from the endoderm to themesoderm, which is critical to gut morphogenesis. Therefore, forexample, hedgehog antagonists of the instant method can be employed forregulating the development and maintenance of an artificial liver thatcan have multiple metabolic functions of a normal liver. In an exemplaryembodiment, the subject method can be used to regulate the proliferationand differentiation of digestive tube stem cells to form hepatocytecultures which can be used to populate extracellular matrices, or whichcan be encapsulated in biocompatible polymers, to form both implantableand extracorporeal artificial livers.

In another embodiment, therapeutic compositions of hedgehog antagonistscan be utilized in conjunction with transplantation of such artificiallivers, as well as embryonic liver structures, to regulate uptake ofintraperitoneal implantation, vascularization, and in vivodifferentiation and maintenance of the engrafted liver tissue.

In yet another embodiment, the subject method can be employedtherapeutically to regulate such organs after physical, chemical orpathological insult. For instance, therapeutic compositions comprisinghedgehog antagonists can be utilized in liver repair subsequent to apartial hepatectomy.

The generation of the pancreas and small intestine from the embryonicgut depends on intercellular signalling between the endodermal andmesodermal cells of the gut. In particular, the differentiation ofintestinal mesoderm into smooth muscle has been suggested to depend onsignals from adjacent endodermal cells. One candidate mediator ofendodermally derived signals in the embryonic hindgut is Sonic hedgehog.See, for example, Apelqvist et al. (1997) Curr Biol 7:801-4. The Shhgene is expressed throughout the embryonic gut endoderm with theexception of the pancreatic bud endoderm, which instead expresses highlevels of the homeodomain protein Ipf1/Pdx1 (insulin promoter factor1/pancreatic and duodenal homeobox 1), an essential regulator of earlypancreatic development. Apelqvist et al., supra, have examined whetherthe differential expression of Shh in the embryonic gut tube controlsthe differentiation of the surrounding mesoderm into specialisedmesoderm derivatives of the small intestine and pancreas. To test this,they used the promoter of the Ipf1/Pdx1 gene to selectively express Shhin the developing pancreatic epithelium. In Ipf1/Pdx1-Shh transgenicmice, the pancreatic mesoderm developed into smooth muscle andinterstitial cells of Cajal, characteristic of the intestine, ratherthan into pancreatic mesenchyme and spleen. Also, pancreatic explantsexposed to Shh underwent a similar program of intestinaldifferentiation. These results provide evidence that the differentialexpression of endodermally derived Shh controls the fate of adjacentmesoderm at different regions of the gut tube.

In the context of the present invention, it is contemplated thereforethat the subject hedgehog antagonists can be used to control or regulatethe proliferation and/or differentiation of pancreatic tissue both invivo and in vitro.

There are a wide variety of pathological cell proliferative anddifferentiative conditions for which the inhibitors of the presentinvention may provide therapeutic benefits, with the general strategybeing, for example, the correction of aberrant insulin expression, ormodulation of differentiation. More generally, however, the presentinvention relates to a method of inducing and/or maintaining adifferentiated state, enhancing survival and/or affecting proliferationof pancreatic cells, by contacting the cells with the subjectinhibitors. For instance, it is contemplated by the invention that, inlight of the apparent involvement of ptc, hedgehog, and smoothened inthe formation of ordered spatial arrangements of pancreatic tissues, thesubject method could be used as part of a technique to generate and/ormaintain such tissue both in vitro and in vivo. For instance, modulationof the function of hedgehog can be employed in both cell culture andtherapeutic methods involving generation and maintenance β-cells andpossibly also for non-pancreatic tissue, such as in controlling thedevelopment and maintenance of tissue from the digestive tract, spleen,lungs, urogenital organs (e.g., bladder), and other organs which derivefrom the primitive gut.

In an exemplary embodiment, the present method can be used in thetreatment of hyperplastic and neoplastic disorders effecting pancreatictissue, particularly those characterized by aberrant proliferation ofpancreatic cells. For instance, pancreatic cancers are marked byabnormal proliferation of pancreatic cells that can result inalterations of insulin secretory capacity of the pancreas For instance,certain pancreatic hyperplasias, such as pancreatic carcinomas, canresult in hypoinsulinemia due to dysfunction of β-cells or decreasedislet cell mass. To the extent that aberrant ptc, hedgehog, andsmoothened signaling may be indicated in disease progression, thesubject inhibitors, can be used to enhance regeneration of the tissueafter anti-tumor therapy.

Moreover, manipulation of hedgehog signaling properties at differentpoints may be useful as part of a strategy for reshaping/repairingpancreatic tissue both in vivo and in vitro. In one embodiment, thepresent invention makes use of the apparent involvement of ptc,hedgehog, and smoothened in regulating the development of pancreatictissue. In general, the subject method can be employed therapeuticallyto regulate the pancreas after physical, chemical or pathologicalinsult. In yet another embodiment, the subject method can be applied toto cell culture techniques, and in particular, may be employed toenhance the initial generation of prosthetic pancreatic tissue devices.Manipulation of proliferation and differentiation of pancreatic tissue,for example, by altering hedgehog activity, can provide a means for morecarefully controlling the characteristics of a cultured tissue. In anexemplary embodiment, the subject method can be used to augmentproduction of prosthetic devices which require β-islet cells, such asmay be used in the encapsulation devices described in, for example, theAebischer et al. U.S. Pat. No. 4,892,538, the Aebischer et al. U.S. Pat.No. 5,106,627, the Lim U.S. Pat. No. 4,391,909, and the Sefton U.S. Pat.No. 4,353,888. Early progenitor cells to the pancreatic islets aremultipotential, and apparently coactivate all the islet-specific genesfrom the time they first appear. As development proceeds, expression ofislet-specific hormones, such as insulin, becomes restricted to thepattern of expression characteristic of mature islet cells. Thephenotype of mature islet cells, however, is not stable in culture, asreappearence of embryonal traits in mature β-celis can be observed. Byutilizing the subject hedgehog antagonists, the differentiation path orproliferative index of the cells can be regulated.

Furthermore, manipulation of the differentiative state of pancreatictissue can be utilized in conjunction with transplantation of artificialpancreas so as to promote implantation, vascularization, and in vivodifferentiation and maintenance of the engrafted tissue. For instance,manipulation of hedgehog function to affect tissue differentiation canbe utilized as a means of maintaining graft viability.

Bellusci et al. (1997) Development 124:53 report that Sonic hedgehogregulates lung mesenchymal cell proliferation in vivo. Accordingly, thepresent method can be used to regulate regeneration of lung tissue,e.g., in the treatment of emphysema and respiratory distress syndrome.

Fujita et al. (1997) Biochem Biophys Res Commun 238:658 reported thatSonic hedgehog is expressed in human lung squamous carcinoma andadenocarcinoma cells. The expression of Sonic hedgehog was also detectedin the human lung squamous carcinoma tissues, but not in the normal lungtissue of the same patient. They also observed that Sonic hedgehogstimulates the incorporation of BrdU into the carcinoma cells andstimulates their cell growth, while anti-Shh-N inhibited their cellgrowth. These results suggest that a ptc, hedgehog, and/or smoothened isinvolved in the cell growth of such transformed lung tissue andtherefore indicates that the subject method can be used as part of atreatment of lung carcinoma and adenocarcinomas, and other proliferativedisorders involving the lung epithelia.

Many other tumors may, based on evidence such as involvement of thehedgehog pathway in these tumors, or detected expression of hedgehog orits receptor in these tissues during development, be affected bytreatment with the subject compounds. Such tumors include, but are by nomeans limited to, tumors related to Gorlin's syndrome (e.g., basal cellcarcinoma, medulloblastoma, meningioma, etc.), tumors evidenced in pctknock-out mice (e.g., hemangioma, rhabdomyosarcoma, etc.), tumorsresulting from gli-1 amplification (e.g., glioblastoma, sarcoma, etc.),tumors connected with TRC8, a ptc homolog (e.g., renal carcinoma,thyroid carcinoma, etc.), Ext-1-related tumors (e.g., bone cancer,etc.), Shh-induced tumors (e.g., lung cancer, chondrosarcomas, etc.),and other tumors (e.g., breast cancer, urogenital cancer (e.g., kidneybladder, ureter, prostate, etc.), fibrosarcoma, hypothalamic hamartoma,adrenal cancer, gastrointestinal cancer (e.g., stomach, intestine(including parts of the small and large intestine), esophagus, and gutannexes such as pancreas, the biliary duct system, etc.), etc.).

In still another embodiment of the present invention, compositionscomprising hedgehog antagonists can be used in the in vitro generationof skeletal tissue, such as from skeletogenic stem cells, as well as thein vivo treatment of skeletal tissue deficiencies. The present inventionparticularly contemplates the use of hedgehog antagonists to regulatethe rate of chondrogenesis and/or osteogenesis. By “skeletal tissuedeficiency”, it is meant a deficiency in bone or other skeletalconnective tissue at any site where it is desired to restore the bone orconnective tissue, no matter how the deficiency originated, e.g. whetheras a result of surgical intervention, removal of tumor, ulceration,implant, fracture, or other traumatic or degenerative conditions.

For instance, the method of the present invention can be used as part ofa regimen for restoring cartilage function to a connective tissue. Suchmethods are useful in, for example, the repair of defects or lesions incartilage tissue which is the result of degenerative wear such as thatwhich results in arthritis, as well as other mechanical derangementswhich may be caused by trauma to the tissue, such as a displacement oftorn meniscus tissue, meniscectomy, a Taxation of a joint by a tornligament, malignment of joints, bone fracture, or by hereditary disease.The present reparative method is also useful for remodeling cartilagematrix, such as in plastic or reconstructive surgery, as well asperiodontal surgery. The present method may also be applied to improvinga previous reparative procedure, for example, following surgical repairof a meniscus, ligament, or cartilage. Furthermore, it may prevent theonset or exacerbation of degenerative disease if applied early enoughafter trauma.

In one embodiment of the present invention, the subject method comprisestreating the afflicted connective tissue with a therapeuticallysufficient amount of a hedgehog antagonist, particularly an antagonistselective for Indian hedgehog signal transduction, to regulate acartilage repair response in the connective tissue by managing the rateof differentiation and/or proliferation of chondrocytes embedded in thetissue. Such connective tissues as articular cartilage, interarticularcartilage (menisci), costal cartilage (connecting the true ribs and thesternum), ligaments, and tendons are particularly amenable to treatmentin reconstructive and/or regenerative therapies using the subjectmethod. As used herein, regenerative therapies include treatment ofdegenerative states which have progressed to the point of whichimpairment of the tissue is obviously manifest, as well as preventivetreatments of tissue where degeneration is in its earliest stages orimminent.

In an illustrative embodiment, the subject method can be used as part ofa therapeutic intervention in the treatment of cartilage of adiarthroidal joint, such as a knee, an ankle, an elbow, a hip, a wrist,a knuckle of either a finger or toe, or a tempomandibular joint. Thetreatment can be directed to the meniscus of the joint, to the articularcartilage of the joint, or both. To further illustrate, the subjectmethod can be used to treat a degenerative disorder of a knee, such aswhich might be the result of traumatic injury (e.g., a sports injury orexcessive wear) or osteoarthritis. The subject antagonists may beadministered as an injection into the joint with, for instance, anarthroscopic needle. In some instances, the injected agent can be in theform of a hydrogel or other slow release vehicle described above inorder to permit a more extended and regular contact of the agent withthe treated tissue.

The present invention further contemplates the use of the subject methodin the field of cartilage transplantation and prosthetic devicetherapies. However, problems arise, for instance, because thecharacteristics of cartilage and fibrocartilage varies between differenttissue such as between articular, meniscal cartilage, ligaments, andtendons, between the two ends of the same ligament or tendon, andbetween the superficial and deep parts of the tissue. The zonalarrangement of these tissues may reflect a gradual change in mechanicalproperties, and failure occurs when implanted tissue, which has notdifferentiated under those conditions, lacks the ability toappropriately respond. For instance, when meniscal cartilage is used torepair anterior cruciate ligaments, the tissue undergoes a metaplasia topure fibrous tissue. By regulating the rate of chondrogenesis, thesubject method can be used to particularly address this problem, byhelping to adaptively control the implanted cells in the new environmentand effectively resemble hypertrophic chondrocytes of an earlierdevelopmental stage of the tissue.

In similar fashion, the subject method can be applied to enhancing boththe generation of prosthetic cartilage devices and to theirimplantation. The need for improved treatment has motivated researchaimed at creating new cartilage that is based oncollagen-glycosaminoglycan templates (Stone et al. (1990) Clin OrthopRelat Red 252:129), isolated chondrocytes (Grande et al. (1989) J OrthopRes 7:208; and Takigawa et al. (1987) Bone Miner 2:449), andchondrocytes attached to natural or synthetic polymers (Walitani et al.(1989) J Bone Jt Surg 71B:74; Vacanti et al. (1991) Plast Reconstr Surg88:753; von Schroeder et al. (1991) J Biomed Mater Res 25:329; Freed etal. (1993) J Biomed Mater Res 27:11; and the Vacanti et al. U.S. Pat.No. 5,041,138). For example, chondrocytes can be grown in culture onbiodegradable, biocompatible highly porous scaffolds formed frompolymers such as polyglycolic acid, polylactic acid, agarose gel, orother polymers that degrade over time as function of hydrolysis of thepolymer backbone into innocuous monomers. The matrices are designed toallow adequate nutrient and gas exchange to the cells until engraftmentoccurs. The cells can be cultured in vitro until adequate cell volumeand density has developed for the cells to be implanted. One advantageof the matrices is that they can be cast or molded into a desired shapeon an individual basis, so that the final product closely resembles thepatient's own ear or nose (by way of example), or flexible matrices canbe used which allow for manipulation at the time of implantation, as ina joint.

In one embodiment of the subject method, the implants are contacted witha hedgehog antagonist during certain stages of the culturing process inorder to manage the rate of differentiation of chondrocytes and theformation of hypeltrophic chrondrocytes in the culture.

In another embodiment, the implanted device is treated with a hedgehogantagonist in order to actively remodel the implanted matrix and to makeit more suitable for its intended function. As set out above withrespect to tissue transplants, the artificial transplants suffer fromthe same deficiency of not being derived in a setting which iscomparable to the actual mechanical environment in which the matrix isimplanted. The ability to regulate the chondrocytes in the matrix by thesubject method can allow the implant to acquire characteristics similarto the tissue for which it is intended to replace.

In yet another embodiment, the subject method is used to enhanceattachment of prosthetic devices. To illustrate, the subject method canbe used in the implantation of a periodontal prosthesis, wherein thetreatment of the surrounding connective tissue stimulates formation ofperiodontal ligament about the prosthesis.

In still further embodiments, the subject method can be employed as partof a regimen for the generation of bone (osteogenesis) at a site in theanimal where such skeletal tissue is deficient. Indian hedgehog isparticularly associated with the hypertrophic chondrocytes that areultimately replaced by osteoblasts. For instance, administration of ahedgehog antagonist of the present invention can be employed as part ofa method for regulating the rate of bone loss in a subject. For example,preparations comprising hedgehog antagonists can be employed, forexample, to control endochondral ossification in the formation of a“model” for ossification.

In yet another embodiment of the present invention, a hedgehogantagonist can be used to regulate spermatogenesis. The hedgehogproteins, particularly Dhh, have been shown to be involved in thedifferentiation and/or proliferation and maintenance of testicular germcells. Dhh expression is initiated in Sertoli cell precursors shortlyafter the activation of Sry (testicular determining gene) and persistsin the testis into the adult. Males are viable but infertile, owing to acomplete absence of mature sperm. Examination of the developing testisin different genetic backgrounds suggests that Dhh regulates both earlyand late stages of spermatogenesis. Bitgood et al. (1996) Curr Biol6:298. In a preferred embodiment, the hedgehog antagonist can be used asa contraceptive. In similar fashion, hedgehog antagonists of the subjectmethod are potentially useful for modulating normal ovarian function.

The subject method also has wide applicability to the treatment orprophylaxis of disorders afflicting epithelial tissue, as well as incosmetic uses. In general, the method can be characterized as includinga step of administering to an animal an amount of a hedgehog antagonisteffective to alter the growth state of a treated epithelial tissue. Themode of administration and dosage regimens will vary depending on theepithelial tissue(s) to be treated. For example, topical formulationswill be preferred where the treated tissue is epidermal tissue, such asdermal or mucosal tissues.

A method that “promotes the healing of a wound” results in the woundhealing more quickly as a result of the treatment than a similar woundheals in the absence of the treatment. “Promotion of wound healing” canalso mean that the method regulates the proliferation and/or growth of,inter alia, keratinocytes, or that the wound heals with less scarring,less wound contraction, less collagen deposition and more superficialsurface area. In certain instances, “promotion of wound healing” canalso mean that certain methods of wound healing have improved successrates, (e.g., the take rates of skin grafts,) when used together withthe method of the present invention.

Despite significant progress in reconstructive surgical techniques,scarring can be an important obstacle in regaining normal function andappearance of healed skin. This is particularly true when pathologicscarring such as keloids or hypertrophic scars of the hands or facecauses functional disability or physical deformity. In the severestcircumstances, such scarring may precipitate psychosocial distress and alife of economic deprivation. Wound repair includes the stages ofhemostasis, inflammation, proliferation, and remodeling. Theproliferative stage involves multiplication of fibroblasts andendothelial and epithelial cells. Through the use of the subject method,the rate of proliferation of epithelial cells in and proximal to thewound can be controlled in order to accelerate closure of the woundand/or minimize the formation of scar tissue.

The present treatment can also be effective as part of a therapeuticregimen for treating oral and paraoral ulcers, e.g. resulting fromradiation and/or chemotherapy. Such ulcers commonly develop within daysafter chemotherapy or radiation therapy. These ulcers usually begin assmall, painful irregularly shaped lesions usually covered by a delicategray necrotic membrane and surrounded by inflammatory tissue. In manyinstances, lack of treatment results in proliferation of tissue aroundthe periphery of the lesion on an inflammatory basis. For instance, theepithelium bordering the ulcer usually demonstrates proliferativeactivity, resulting in loss of continuity of surface epithelium. Theselesions, because of their size and loss of epithelial integrity, disposethe body to potential secondary infection. Routine ingestion of food andwater becomes a very painful event and, if the ulcers proliferatethroughout the alimentary canal, diarrhea usually is evident with allits complicating factors. According to the present invention, atreatment for such ulcers which includes application of a hedgehogantagonist can reduce the abnormal proliferation and differentiation ofthe affected epithelium, helping to reduce the severity of subsequentinflammatory events.

The subject method and compositions can also be used to treat woundsresulting from dermatological diseases, such as lesions resulting fromautoimmune disorders such as psoriasis. Atopic dermititis refers to skintrauma resulting from allergies associated with an immune responsecaused by allergens such as pollens, foods, dander, insect venoms andplant toxins.

In other embodiments, antiproliferative preparations of hedgehogantagonists can be used to inhibit lens epithelial cell proliferation toprevent post-operative complications of extracapsular cataractextraction. Cataract is an intractable eye disease and various studieson a treatment of cataract have been made. But at present, the treatmentof cataract is attained by surgical operations. Cataract surgery hasbeen applied for a long time and various operative methods have beenexamined. Extracapsular lens extraction has become the method of choicefor removing cataracts. The major medical advantages of this techniqueover intracapsular extraction are lower incidence of aphakic cystoidmacular edema and retinal detachment. Extracapsular extraction is alsorequired for implantation of posterior chamber type intraocular lensesthat are now considered to be the lenses of choice in most cases.

However, a disadvantage of extracapsular cataract extraction is the highincidence of posterior lens capsule opacification, often calledafter-cataract, which can occur in up to 50% of cases within three yearsafter surgery. After-cataract is caused by proliferation of equatorialand anterior capsule lens epithelial cells that remain afterextracapsular lens extraction. These cells proliferate to causeSommerling rings, and along with fibroblasts that also deposit and occuron the posterior capsule, cause opacification of the posterior capsule,which interferes with vision. Prevention of after-cataract would bepreferable to treatment. To inhibit secondary cataract formation, thesubject method provides a means for inhibiting proliferation of theremaining lens epithelial cells. For example, such cells can be inducedto remain quiescent by instilling a solution containing an hedgehogantagonist preparation into the anterior chamber of the eye after lensremoval. Furthermore, the solution can be osmotically balanced toprovide minimal effective dosage when instilled into the anteriorchamber of the eye, thereby inhibiting subcapsular epithelial growthwith some specificity.

The subject method can also be used in the treatment of comeopathiesmarked by corneal epithelial cell proliferation, as for example inocular epithelial disorders such as epithelial downgrowth or squamouscell carcinomas of the ocular surface.

Levine et al. (1997) J Neurosci 17:6277 show that hedgehog proteins canregulate mitogenesis and photoreceptor differentiation in the vertebrateretina, and Ihh is a candidate factor from the pigmented epithlium topromote retinal progenitor proliferation and photoreceptordifferentiation. Likewise, Jensen et al. (1997) Development 124:363demonstrated that treatment of cultures of perinatal mouse retinal cellswith the amino-terminal fragment of Sonic hedgehog protein results in anincrease in the proportion of cells that incorporate bromodeoxuridine,in total cell numbers, and in rod photoreceptors, amacrine cells andMuller glial cells, suggesting that Sonic hedgehog promotes theproliferation of retinal precursor cells. Thus, the subject method canbe used in the treatment of proliferative diseases of retinal cells andregulate photoreceptor differentiation.

Yet another aspect of the present invention relates to the use of thesubject method to control hair growth. Hair is basically composed ofkeratin, a tough and insoluble protein; its chief strength lies in itsdisulphide bond of cystine. Each individual hair comprises a cylindricalshaft and a root, and is contained in a follicle, a flask-likedepression in the skin. The bottom of the follicle contains afinger-like projection termed the papilla, which consists of connectivetissue from which hair grows, and through which blood vessels supply thecells with nourishment. The shaft is the part that extends outwards fromthe skin surface, whilst the root has been described as the buried partof the hair. The base of the root expands into the hair bulb, whichrests upon the papilla. Cells from which the hair is produced grow inthe bulb of the follicle; they are extruded in the form of fibers as thecells proliferate in the follicle. Hair “growth” refers to the formationand elongation of the hair fiber by the dividing cells.

As is well known in the art, the common hair cycle is divided into threestages: anagen, catagen and telogen. During the active phase (anagen),the epidermal stem cells of the dermal papilla divide rapidly. Daughtercells move upward and differentiate to form the concentric layers of thehair itself. The transitional stage, catagen, is marked by the cessationof mitosis of the stem cells in the follicle. The resting stage is knownas telogen, where the hair is retained within the scalp for severalweeks before an emerging new hair developing below it dislodges thetelogen-phase shaft from its follicle. From this model it has becomeclear that the larger the pool of dividing stem cells that differentiateinto hair cells, the more hair growth occurs. Accordingly, methods forincreasing or reducing hair growth can be carried out by potentiating orinhibiting, respectively, the proliferation of these stein cells.

In certain embodiments, the subject method can be employed as a way ofreducing the growth of human hair as opposed to its conventional removalby cutting, shaving, or depilation. For instance, the present method canbe used in the treatment of trichosis characterized by abnormally rapidor dense growth of hair, e.g. hypertrichosis. In an exemplaryembodiment, hedgehog antagonists can be used to manage hirsutism, adisorder marked by abnormal hairiness. The subject method can alsoprovide a process for extending the duration of depilation.

Moreover, because a hedgehog antagonist will often be cytostatic toepithelial cells, rather than cytotoxic, such agents can be used toprotect hair follicle cells from cytotoxic agents that requireprogression into S-phase of the cell-cycle for efficacy, e.g.radiation-induced death. Treatment by the subject method can provideprotection by causing the hair follicle cells to become quiescent, e.g.,by inhibiting the cells from entering S phase, and thereby preventingthe follicle cells from undergoing mitotic catastrophe or programmedcell death. For instance, hedgehog antagonists can be used for patientsundergoing chemo- or radiation-therapies that ordinarily result in hairloss. By inhibiting cell-cycle progression during such therapies, thesubject treatment can protect hair follicle cells from death that mightotherwise result from activation of cell death programs. After thetherapy has concluded, the instant method can also be removed withconcommitant relief of the inhibition of follicle cell proliferation.

The subject method can also be used in the treatment of folliculitis,such as folliculitis decalvans, folliculitis ulerythematosa reticulataor keloid folliculitis. For example, a cosmetic prepration of anhedgehog antagonist can be applied topically in the treatment ofpseudofolliculitis, a chronic disorder occurring most often in thesubmandibular region of the neck and associated with shaving, thecharacteristic lesions of which are erythematous papules and pustulescontaining buried hairs.

In another aspect of the invention the subject method can be usd toinduce differentiation and/or inhibit proliferation of epitheliallyderived tissue. Such forms of these molecules can provide a basis fordifferentiation therapy for the treatment of hyperplastic and/orneoplastic conditions involving epithelial tissue. For example, suchpreparations can be used for the treatment of cutaneous diseases inwhich there is abnormal proliferation or growth of cells of the skin.

For instance, the pharmaceutical preparations of the invention areintended for the treatment of hyperplastic epidermal conditions, such askeratosis, as well as for the treatment of neoplastic epidermalconditions such as those characterized by a high proliferation rate forvarious skin cancers, as for example basal cell carcinoma or squamouscell carcinoma. The subject method can also be used in the treatment ofautoimmune diseases affecting the skin, in particular, of dermatologicaldiseases involving morbid proliferation and/or keratinization of theepidermis, as for example, caused by psoriasis or atopic dermatosis.

Many common diseases of the skin, such as psoriasis, squamous cellcarcinoma, keratoacanthoma and actinic keratosis are characterized bylocalized abnormal proliferation and growth. For example, in psoriasis,which is characterized by scaly, red, elevated plaques on the skin, thekeratinocytes are known to proliferate much more rapidly than normal andto differentiate less completely.

In one embodiment, the preparations of the present invention aresuitable for the treatment of dermatological ailments linked tokeratinization disorders causing abnormal proliferation of skin cells,which disorders may be marked by either inflammatory or non-inflammatorycomponents. To illustrate, therapeutic preparations of a hedgehogantagonist, e.g., which promotes quiescense or differentiation can beused to treat varying forms of psoriasis, be they cutaneous, mucosal orungual. Psoriasis, as described above, is typically characterized byepidermal keratinocytes that display marked proliferative activation anddifferentiation along a “regenerative” pathway. Treatment with anantiproliferative embodiment of the subject method can be used toreverse the pathological epidermal activiation and can provide a basisfor sustained remission of the disease.

A variety of other keratotic lesions are also candidates for treatmentwith the subject method. Actinic keratoses, for example, are superficialinflammatory premalignant tumors arising on sun-exposed and irradiatedskin. The lesions are erythematous to brown with variable scaling.Current therapies include excisional and cryosurgery. These treatmentsare painfil, however, and often produce cosmetically unacceptablescarring. Accordingly, treatment of keratosis, such as actinickeratosis, can include application, preferably topical, of a hedgehogantagonist composition in amounts sufficient to inhibithyperproliferation of epidermal/epidermoid cells of the lesion.

Acne represents yet another dermatologic ailment which may be treated bythe subject method. Acne vulgaris, for instance, is a multifactorialdisease most commonly occurring in teenagers and young adults, and ischaracterized by the appearance of inflammatory and noninflammatorylesions on the face and upper trunk. The basic defect which gives riseto acne vulgaris is hypercornification of the duct of a hyperactivesebaceous gland. Hypercornification blocks the normal mobility of skinand follicle microorganisms, and in so doing, stimulates the release oflipases by Propinobacterium acnes and Staphylococcus epidermidisbacteria and Pitrosporum ovate, a yeast. Treatment with anantiproliferative hedgehog antagonist, particularly topicalpreparations, may be useful for preventing the transitional features ofthe ducts, e.g. hypercornification, which lead to lesion formation. Thesubject treatment may further include, for example, antibiotics,retinoids and antiandrogens.

The present invention also provides a method for treating various formsof dermatitis. Dermatitis is a descriptive term referring to poorlydemarcated lesions that are either pruritic, erythematous, scaley,blistered, weeping, fissured or crusted. These lesions arise from any ofa wide variety of causes. The most common types of dermatitis areatopic, contact and diaper dermatitis. For instance, seborrheicdermatitis is a chronic, usually pruritic, dermatitis with erythema,dry, moist, or greasy scaling, and yellow crusted patches on variousareas, especially the scalp, with exfoliation of an excessive amount ofdry scales. The subject method can also be used in the treatment ofstasis dermatitis, an often chronic, usually eczematous dermatitis.Actinic dermatitis is dermatitis that due to exposure to actinicradiation such as that from the sun, ultraviolet waves or x- orgamma-radiation. According to the present invention, the subject methodcan be used in the treatment and/or prevention of certain symptoms ofdermatitis caused by unwanted proliferation of epithelial cells. Suchtherapies for these various forms of dermatitis can also include topicaland systemic corticosteroids, antipuritics, and antibiotics.

For example, it is contemplated that the subject method could be used toinhibit angiogenesis. Hedgehog is known to stimulate angiogenesis.Matrigel plugs impregnated with hedgehog protein and inserted into miceevince substantial neovascularization, whereas Matrigel plugs notcarrying hedgehog show comparatively little vascularization. Hedgehogprotein is also capable of increasing vascularization of the normallyavascular mouse cornea. The pct-1 gene is expressed in normal vasculartissues, including the endothelial cells of the aorta, vascular smoothmuscle cells, adventitial fibroblasts of the aorta, the coronaryvasculature and cardiomyocytes of the atria and ventricles. Thesetissues are also sensitive to hedgehog protein. Treatment with exogenoushedgehog causes upregulation of ptc-1 expression. In addition, hedgehogproteins stimulate proliferation of vascular smooth muscle cells invivo. Hedgehog proteins also cause fibroblasts to increase expression ofangiogenic growth factors such as VEGF, bFGF, Ang-1 and Ang-2. Lastly,hedgehog proteins are known to stimulate recovery from ischemic injuryand stimulate formation of collateral vessels.

Given that hedgehog promotes angiogenesis, hedgehog antagonists areexpected to act as angiogenesis inhibitors, particularly in situationswhere some level of hedgehog signaling is necessary for angiogenesis.

Angiogenesis is fundamental to many disorders. Persistent, unregulatedangiogenesis occurs in a range of disease states, tumor metastases andabnormal growths by endothelial cells. The vasculature created as aresult of angiogenic processes supports the pathological damage seen inthese conditions. The diverse pathological states created due tounregulated angiogenesis have been grouped together as angiogenicdependent or angiogenic associated diseases. Therapies directed atcontrol of the angiogenic processes could lead to the abrogation ormitigation of these diseases.

Diseases caused by, supported by or associated with angiogenesis includeocular neovascular disease, age-related macular degeneration, diabeticretinopathy, retinopathy of prematurity, corneal graft rejection,neovascular glaucoma, retrolental fibroplasia, epidemickeratoconjunctivitis, Vitamin A deficiency, contact lens overwear,atopic keratitis, superior limbic keratitis, pterygium keratitis sicca,Sjogren's, acne rosacea, phylectenulosis, syphilis, Mycobacteriainfections, lipid degeneration, chemical burns, bacterial ulcers, fungalulcers, Herpes simplex infections, Herpes zoster infections, protozoaninfections, Kaposi sarcoma, Mooren ulcer, Terrien's marginaldegeneration, marginal keratolysis, rheumatoid arthritis, systemiclupus, polyarteritis, trauma, Wegeners sarcoidosis, Scleritis, StevensJohnson disease, periphigoid radial keratotomy, corneal graph rejection,rheumatoid arthritis, osteoarthritis chronic inflammation (eg.,ulcerative colitis or Crohn's disease), hemangioma, Osler-Weber-Rendudisease, and hereditary hemorrhagic telangiectasia.

In addition, angiogenesis plays a critical role in cancer. A tumorcannot expand without a blood supply to provide nutrients and removecellular wastes. Tumors in which angiogenesis is important include solidtumors such as rhabdomyosarcomas, retinoblastoma, Ewing sarcoma,neuroblastoma, and osteosarcoma, and benign tumors such as acousticneuroma, neurofibroma, trachoma and pyogenic granulomas. Angiogenicfactors have been found associated with several solid tumors. Preventionof angiogenesis could halt the growth of these tumors and the resultantdamage to the animal due to the presence of the tumor. Angiogenesis isalso associated with blood-born tumors such as leukemias, any of variousacute or chronic neoplastic diseases of the bone marrow in whichunrestrained proliferation of white blood cells occurs, usuallyaccompanied by anemia, impaired blood clotting, and enlargement of thelymph nodes, liver, and spleen. It is believed that angiogenesis plays arole in the abnormalities in the bone marrow that give rise toleukemia-like tumors.

In addition to tumor growth, angiogensis is imortant in metastasis.Initially, angiogenesis is important is in the vascularization of thetumor which allows cancerous cells to enter the blood stream and tocirculate throughout the body. After the tumor cells have left theprimary site, and have settled into the secondary, metastasis site,angiogenesis must occur before the new tumor can grow and expand.Therefore, prevention of angiogenesis could lead to the prevention ofmetastasis of tumors and possibly contain the neoplastic growth at theprimary site.

Angiogenesis is also involved in normal physiological processes such asreproduction and wound healing. Angiogenesis is an important step inovulation and also in implantation of the blastula after fertilization.Prevention of angiogenesis could be used to induce amenorrhea, to blockovulation or to prevent implantation by the blastula.

It is anticipated that the invention will be useful for the treatmentand/or prevention of respiratory distress syndrome or other disordersresulting from inappropriate lung surface tension. Respiratory distresssyndrome results from insufficient surfactant in the alveolae of thelungs. The lungs of vertebrates contain surfactant, a complex mixture oflipids and protein that causes surface tension to rise during lunginflation and decrease during lung deflation. During lung deflation,surfactant decreases such that there are no surface forces that wouldotherwise promote alveolar collapse. Aerated alveoli that have notcollapsed during expiration permit continuous oxygen and carbon dioxidetransport between blood and alveolar gas and require much less force toinflate during the subsequent inspiration. During inflation, lungsurfactant increases surface tension as the alveolar surface areaincreases. A rising surface tension in expanding alveoli opposesover-inflation in those airspaces and tends to divert inspired air toless well-aerated alveoli, thereby facilitating even lung aeration.

Respiratory distress syndrome is particularly prevalent among prematureinfants. Lung surfactant is normally synthesized at a very low rateuntil the last six weeks of fetal life. Human infants born more than sixweeks before the normal term of a pregnancy have a high risk of beingborn with inadequate amounts of lung surfactant and inadequate rates ofsurfactant synthesis. The more prematurely an infant is born, the moresevere the surfactant deficiency is likely to be. Severe surfactantdeficiency can lead to respiratory failure within a few minutes or hoursof birth. The surfactant deficiency produces progressive collapse ofalveoli (atelectasis) because of the decreasing ability of the lung toexpand despite maximum inspiratory effort. As a result, inadequateamounts of oxygen reach the infant's blood. RDS can occur in adults aswell, typically as a consequence of failure in surfactant biosynthesis.

Lung tissue of premature infants shows high activity of the hedgehogsignaling pathway. Inhibition of this pathway using hedgehog antagonistsincreases the formation of lamellar bodies and increases the expressionof genes involved in surfactant biosynthesis. Lamellar bodies aresubcellular structures associated with surfactant biosynthesis. Forthese reasons, treatment of premature infants with a hedgehog antagonistshould stimulate surfactant biosynthesis and ameliorate RDS. In caseswhere adult RDS is associated with hedgehog pathway activation,treatment with hedgehog antagonists should also be effective.

It is further contemplated that the use of hedgehog antagonists may bespecifically targeted to disorders where the affected tissue and/orcells evince high hedgehog pathway activation. Expression of gli genesis activated by the hedgehog signaling pathway, including gli-1, gli-2and gli-3. gli-1 expression is most consistently correlated withhedgehog signaling activity across a wide range of tissues anddisorders, while gli-3 is somewhat less so. The gli genes encodetranscription factors that activate expression of many genes needed toelicit the full effects of hedgehog signaling. However, the Gli-3transcription factor can also act as a repressor of hedgehog effectorgenes, and therefore, expression of gli-3 can cause a decreased effectof the hedgehog signaling pathway. Whether Gli-3 acts as atranscriptional activator or repressor depends on post-translationalevents, and therefore it is expected that methods for detecting theactivating form (versus the repressing form) of Gli-3 protein would alsobe a reliable measure of hedgehog pathway activation. gli-2 geneexpression is expected to provide a reliable marker for hedgehog pathwayactivation. The gli-1 gene is strongly expressed in a wide array ofcancers, hyperplasias and immature lungs, and serves as a marker for therelative activation of the hedgehog pathway. In addition, tissues, suchas immature lung, that have high gli gene expression are stronglyaffected by hedgehog inhibitors. Accordingly, it is contemplated thatthe detection of gli gene expression may be used as a powerfulpredictive tool to identify tissues and disorders that will particularlybenefit from treatment with a hedgehog antagonist.

In preferred embodiments, gli-1 expression levels are detected, eitherby direct detection of the transcript or by detection of protein levelsor activity. Transcripts may be detected using any of a wide range oftechniques that depend primarily on hybridization of probes to the gli-1transcripts or to cDNAs synthesized therefrom. Well known techniquesinclude Northern blotting, reverse-transcriptase PCR and microarrayanalysis of transcript levels. Methods for detecting Gli protein levelsinclude Western blotting, immunoprecipitation, two-dimensionalpolyacrylamide gel electrophoresis (2D SDS-PAGE)(preferably comparedagainst a standard wherein the position of the Gli proteins has beendetermined), and mass spectroscopy. Mass spectroscopy may be coupledwith a series of purification steps to allow high-throughputidentification of many different protein levels in a particular sample.Mass spectroscopy and 2D SDS-PAGE can also be used to identifypost-transcriptional modifications to proteins including proteolyticevents, ubiquitination, phosphorylation, lipid modification etc. Gliactivity may also be assessed by analyzing binding to substrate DNA orin vitro transcriptional activation of target promoters. Gel shiftassays, DNA footprinting assays and DNA-protein crosslinking assays areall methods that may be used to assess the presence of a protein capableof binding to Gli binding sites on DNA.

In preferred embodiments, gli transcript levels are measured anddiseased or disordered tissues showing abnormally high gli levels aretreated with a hedgehog antagonist. Premature lung tissue, lung cancers(e.g., adenocarcinomas, bronchoalveolar adenocarcinomas, small cellcarcinomas), breast cancers (e.g., inferior ductal carcinomas, inferiorlobular carcinomas, tubular carcinomas), prostate cancers (e.g.,adenocarcinomas), and benign prostatic hyperplasias all show stronglyelevated gli-1 expression levels in certain cases. Accordingly, gli-1expression levels are a powerful diagnostic device to determine which ofthese tissues should be treated with a hedgehog antagonist. In addition,there is substantial correlative evidence that cancers of urothelialcells (e.g., bladder cancer, other urogenital cancers) will also haveelevated gli-1 levels in certain cases. For example, it is known thatloss of heterozygosity on chromosome 9q22 is common in bladder cancers.The ptc-1 gene is located at this position and ptc-1 loss of function isprobably a partial cause of hyperproliferation, as in many other cancertypes. Accordingly, such cancers would also show high gli expression andwould be particularly amenable to treatment with a hedgehog antagonist.

Expression of ptc-1 and ptc-2 is also activated by the hedgehogsignaling pathway, but these genes are inferior to the gli genes asmarkers of hedgehog pathway activation. In certain tissues only one ofptc-1 or ptc-2 is expressed although the hedgehog pathway is highlyactive. For example, in testicular development, Indian hedgehog plays animportant role and the hedgehog pathway is activated, but onlyptc-2 isexpressed. Accordingly, these genes are individually unreliable asmarkers for hedgehog pathway activation, although simultaneousmeasurement of both genes are contemplated as a useful indicator fortissues to be treated with a hedgehog antagonist.

Ailments which may be treated by the subject method are disordersspecific to non-humans, such as mange.

In still another embodiment, the subject method can be used in thetreatment of human cancers, particularly basal cell carcinomas and othertumors of epithelial tissues such as the skin. For example, hedgehogantagonists can be employed, in the subject method, as part of atreatment for basal cell nevus syndrome (BCNS), and other other humancarcinomas, adenocarcinomas, sarcomas and the like.

In a preferred embodiment, the subject method is used as part of atreatment or prophylaxis regimen for treating (or preventing) basal cellcarcinoma. The deregulation of the hedgehog signaling pathway may be ageneral feature of basal cell carcinomas caused by ptc mutations.Consistent overexpression of human ptc mRNA has been described in tumorsof familial and sporadic BCCs, determined by in situ hybridization.Mutations that inactivate ptc may be expected to result inoverexpression of mutant Ptc, because ptc displays negativeautoregulation. Prior research demonstrates that overexpression ofhedgehog proteins can also lead to tumorigenesis. That sonic hedgehog(Shh) has a role in tumorigenesis in the mouse has been suggested byresearch in which transgenic mice overexpressing Shh in the skindeveloped features of BCNS, including multiple BCC-like epidermalproliferations over the entire skin surface, after only a few days ofskin development. A mutation in the Shh human gene from a BCC was alsodescribed; it was suggested that Shh or other Hh genes in humans couldact as dominant oncogenes in humans. Sporadic ptc mutations have alsobeen observed in BCCs from otherwise normal individuals, some of whichare UV-signature mutations. In one recent study of sporadic BCCs, fiveUV-signature type mutations, either CT or CCTT changes, were found outof fifteen tumors determined to contain ptc mutations. Another recentanalysis of sporadic ptc mutations in BCCs and neuroectodermal tumorsrevealed one CT change in one of three ptc mutations found in the BCCs.See, for example, Goodrich et al. (1997) Science 277:1109-13; Xie et al.(1997) Cancer Res 57:2369-72; Oro et al. (1997) Science 276:817-21; Xieet al. (1997) Genes Chromosomes Cancer 18:305-9; Stone et al. (1996)Nature 384:129-34; and Johnson et al. (1996) Science 272:1668-71.

The subject method can also be used to treatment patients with BCNS,e.g., to prevent BCC or other effects of the disease which may be theresult of ptc loss-of-function, hedgehog gain-of-function, or smoothenedgain-of-function. Basal cell nevus syndrome is a rare autosomal dominantdisorder characterized by multiple BCCs that appear at a young age. BCNSpatients are very susceptible to the development of these tumors; in thesecond decade of life, large numbers appear, mainly on sun-exposed areasof the skin. This disease also causes a number of developmentalabnormalities, including rib, head and face alterations, and sometimespolydactyly, syndactyly, and spina bifida. They also develop a number oftumor types in addition to BCCs: fibromas of the ovaries and heart,cysts of the skin and jaws, and in the central nervous system,medulloblastomas and meningiomas. The subject method can be used toprevent or treat such tumor types in BCNS and non-BCNS patients. Studiesof BCNS patients show that they have both genomic and sporadic mutationsin the ptc gene, suggesting that these mutations are the ultimate causeof this disease.

In another aspect, the present invention provides pharmaceuticalpreparations comprising hedgehog antagonists. The hedgehog antagonistsfor use in the subject method may be conveniently formulated foradministration with a biologically acceptable medium, such as water,buffered saline, polyol (for example, glycerol, propylene glycol, liquidpolyethylene glycol and the like) or suitable mixtures thereof. Theoptimum concentration of the active ingredient(s) in the chosen mediumcan be determined empirically, according to procedures well known tomedicinal chemists. As used herein, “biologically acceptable medium”includes any and all solvents, dispersion media, and the like which maybe appropriate for the desired route of administration of thepharmaceutical preparation. The use of such media for pharmaceuticallyactive substances is known in the art. Except insofar as anyconventional media or agent is incompatible with the activity of thehedgehog antagonist, its use in the pharmaceutical preparation of theinvention is contemplated. Suitable vehicles and their formulationinclusive of other proteins are described, for example, in the bookRemington's Pharmaceutical Sciences (Remington's PharmaceuticalSciences. Mack Publishing Company, Easton, Pa., USA 1985). Thesevehicles include injectable “deposit formulations”.

Pharmaceutical formulations of the present invention can also includeveterinary compositions, e.g., pharmaceutical preparations of thehedgehog antagonists suitable for veterinary uses, e.g., for thetreatment of livestock or domestic animals, e.g., dogs.

Methods of introduction may also be provided by rechargeable orbiodegradable devices. Various slow release polymeric devices have beendeveloped and tested in vivo in recent years for the controlled deliveryof drugs, including proteinacious biopharmaceuticals. A variety ofbiocompatible polymers (including hydrogels), including bothbiodegradable and non-degradable polymers, can be used to form animplant for the sustained release of a hedgehog antagonist at aparticular target site.

The preparations of the present invention may be given orally,parenterally, topically, or rectally. They are of course given by formssuitable for each adiministration route. For example, they areadministered in tablets or capsule form, by injection, inhalation, eyelotion, ointment, suppository, controlled release patch, etc.administration by injection, infusion or inhalation; topical by lotionor ointment; and rectal by suppositories. Oral and topicaladministrations are preferred.

The phrases “parenteral administration” and “administered parenterally”as used herein means modes of administration other than enteral andtopical administration, usually by injection, and includes, withoutlimitation, intravenous, intramuscular, intraarterial, intrathecal,intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal,transtracheal, subcutaneous, subcuticular, intraarticulare, subcapsular,subarachnoid, intraspinal and intrastemal injection and infusion.

The phrases “systemic administration,” “administered systemically,”“peripheral administration” and “administered peripherally” as usedherein mean the administration of a compound, drug or other materialother than directly into the central nervous system, such that it entersthe patient's system and, thus, is subject to metabolism and other likeprocesses, for example, subcutaneous administration.

These compounds may be administered to humans and other animals fortherapy by any suitable route of administration, including orally,nasally, as by, for example, a spray, rectally, intravaginally,parenterally, intracistemally and topically, as by powders, ointments ordrops, including buccally and sublingually.

Regardless of the route of administration selected, the compounds of thepresent invention, which may be used in a suitable hydrated form, and/orthe pharmaceutical compositions of the present invention, are formulatedinto pharmaceutically acceptable dosage forms such as described below orby other conventional methods known to those of skill in the art.

Actual dosage levels of the active ingredients in the pharmaceuticalcompositions of this invention may be varied so as to obtain an amountof the active ingrediant that is effective to achieve the desiredtherapeutic reponse for a particular patient, composition, and mode ofadministration, without being toxic to the patient.

The selected dosage level will depend upon a variety of factorsincluding the activity of the particular compound of the presentinvention employed, or the ester, salt or amide thereof, the route ofadministration, the time of administration, the rate of excretion of theparticular compound being employed, the duration of the treatment, otherdrugs, compounds and/or materials used in combination with theparticular hedgehog antagonist employed, the age, sex, weight,condition, general health and prior medical history of the patient beingtreated, and like factors well known in the medical arts.

A physician or veterinarian having ordinary skill in the art can readilydetermine and prescribe the effective amount of the pharmaceuticalcomposition required. For example, the physician or veterinarian couldstart doses of the compounds of the invention employed in thepharmaceutical composition at levels lower than that required in orderto achieve the desired therapeutic effect and gradually increase thedosage until the desired effect is achieved.

In general, a suitable daily dose of a compound of the invention will bethat amount of the compound that is the lowest dose effective to producea therapeutic effect. Such an effective dose will generally depend uponthe factors described above. Generally, intravenous,intracerebroventricular and subcutaneous doses of the compounds of thisinvention for a patient will range from about 0.0001 to about 100 mg perkilogram of body weight per day.

If desired, the effective daily dose of the active compound may beadministered as two, three, four, five, six or more sub-dosesadministered separately at appropriate intervals throughout the day,optionally, in unit dosage forms.

The term “treatment” is intended to encompass also prophylaxis, therapyand cure.

The patient receiving this treatment is any animal in need, includingprimates, in particular humans, and other mammals such as equines,cattle, swine and sheep; and poultry and pets in general.

The compound of the invention can be administered as such or inadmixtures with pharmaceutically acceptable and/or sterile carriers andcan also be administered in conjunction with other antimicrobial agentssuch as penicillins, cephalosporins, aminoglycosides and glycopeptides.Conjunctive therapy, thus includes sequential, simultaneous and separateadministration of the active compound in a way that the therapeuticaleffects of the first administered have not entirely disappeared when thesubsequent agent is administered.

V. Pharmaceutical Compositions

While it is possible for a compound of the present invention to beadministered alone, it is preferable to administer the compound as apharmaceutical formulation (composition). The hedgehog antagonistsaccording to the invention may be formulated for administration in anyconvenient way for use in human or veterinary medicine. In certainembodiments, the compound included in the pharmaceutical preparation maybe active itself, or may be a prodrug, e.g., capable of being convertedto an active compound in a physiological setting.

Thus, another aspect of the present invention provides pharmaceuticallyacceptable compositions comprising a therapeutically effective amount ofone or more of the compounds described above, formulated together withone or more pharmaceutically acceptable carriers (additives) and/ordiluents. As described in detail below, the pharmaceutical compositionsof the present invention may be specially formulated for administrationin solid or liquid form, including those adapted for the following: (1)oral administration, for example, drenches (aqueous or non-aqueoussolutions or suspensions), tablets, boluses, powders, granules, pastesfor application to the tongue; (2) parenteral administration, forexample, by subcutaneous, intramuscular or intravenous injection as, forexample, a sterile solution or suspension; (3) topical application, forexample, as a cream, ointment or spray applied to the skin; or (4)intravaginally or intrarectally, for example, as a pessary, cream orfoam. However, in certain embodiments the subject compounds may besimply dissolved or suspended in sterile water. In certain embodiments,the pharmaceutical preparation is non-pyrogenic, i.e., does not elevatethe body temperature of a patient.

The phrase “therapeutically effective amount” as used herein means thatamount of a compound, material, or composition comprising a compound ofthe present invention which is effective for producing some desiredtherapeutic effect by overcoming a ptc loss-of-function, hedgehoggain-of-function, or smoothened gain-of-function in at least asub-population of cells in an animal and thereby blocking the biologicalconsequences of that pathway in the treated cells, at a reasonablebenefit/risk ratio applicable to any medical treatment.

The phrase “pharmaceutically acceptable” is employed herein to refer tothose compounds, materials, compositions, and/or dosage forms which are,within the scope of sound medical judgment, suitable for use in contactwith the tissues of human beings and animals without excessive toxicity,irritation, allergic response, or other problem or complication,commensurate with a reasonable benefit/risk ratio.

The phrase “pharmaceutically acceptable carrier” as used herein means apharmaceutically acceptable material, composition or vehicle, such as aliquid or solid filler, diluent, excipient, solvent or encapsulatingmaterial, involved in carrying or transporting the subject antagonistsfrom one organ, or portion of the body, to another organ, or portion ofthe body. Each carrier must be “acceptable” in the sense of beingcompatible with the other ingredients of the formulation and notinjurious to the patient. Some examples of materials which can serve aspharmaceutically acceptable carriers include: (1) sugars, such aslactose, glucose and sucrose; (2) starches, such as corn starch andpotato starch; (3) cellulose, and its derivatives, such as sodiumcarboxymethyl cellulose, ethyl cellulose and cellulose acetate; (4)powdered tragacanth; (5) malt; (6) gelatin; (7) talc; (8) excipients,such as cocoa butter and suppository waxes; (9) oils, such as peanutoil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil andsoybean oil; (10) glycols, such as propylene glycol; (11) polyols, suchas glycerin, sorbitol, mannitol and polyethylene glycol; (12) esters,such as ethyl oleate and ethyl laurate; (13) agar; (14) bufferingagents, such as magnesium hydroxide and aluminum hydroxide; (15) alginicacid; (16) pyrogen-free water; (17) isotonic saline; (18) Ringer'ssolution; (19) ethyl alcohol; (20) phosphate buffer solutions; and (21)other non-toxic compatible substances employed in pharmaceuticalformulations.

As set out above, certain embodiments of the present hedgehogantagonists may contain a basic functional group, such as amino oralkylamino, and are, thus, capable of forming pharmaceuticallyacceptable salts with pharmaceutically acceptable acids. The term“pharmaceutically acceptable salts” in this respect, refers to therelatively non-toxic, inorganic and organic acid addition salts ofcompounds of the present invention. These salts can be prepared in situduring the final isolation and purification of the compounds of theinvention, or by separately reacting a purified compound of theinvention in its free base form with a suitable organic or inorganicacid, and isolating the salt thus formed. Representative salts includethe hydrobromide, hydrochloride, sulfate, bisulfate, phosphate, nitrate,acetate, valerate, oleate, palmitate, stearate, laurate, benzoate,lactate, phosphate, tosylate, citrate, maleate, fumarate, succinate,tartrate, napthylate, mesylate, glucoheptonate, lactobionate, andlaurylsulphonate salts and the like. (See, for example, Berge et al.(1977) “Pharmaceutical Salts”, J. Pharm. Sci. 66:1-19)

The pharmaceutically acceptable salts of the subject compounds includethe conventional nontoxic salts or quaternary ammonium salts of thecompounds, e.g., from non-toxic organic or inorganic acids. For example,such conventional nontoxic salts include those derived from inorganicacids such as hydrochloride, hydrobromic, sulfuiric, sulfamic,phosphoric, nitric, and the like; and the salts prepared from organicacids such as acetic, propionic, succinic, glycolic, stearic, lactic,malic, tartaric, citric, ascorbic, palmitic, maleic, hydroxymaleic,phenylacetic, glutamic, benzoic, salicyclic, sulfanilic,2-acetoxybenzoic, fumaric, toluenesulfonic, methanesulfonic, ethanedisulfonic, oxalic, isothionic, and the like.

In other cases, the compounds of the present invention may contain oneor more acidic functional groups and, thus, are capable of formingpharmaceutically acceptable salts with pharmaceutically acceptablebases. The term “pharmaceutically acceptable salts” in these instancesrefers to the relative non-toxic, inorganic and organic base additionsalts of compounds of the present invention. These salts can likewise beprepared in situ during the final isolation and purification of thecompounds, or by separately reacting the purified compound in its freeacid form with a suitable base, such as the hydroxide, carbonate orbicarbonate of a pharmaceutically acceptable metal cation, with ammonia,or with a pharmaceutically acceptable organic primary, secondary ortertiary amine. Representative alkali or alkaline earth salts includethe lithium, sodium, potassium, calcium, magnesium, and aluminum saltsand the like. Representative organic amines useful for the formation ofbase addition salts include ethylamine, diethylamine, ethylenediamine,ethanolamine, diethanolamine, piperazine and the like. (See, forexample, Berge et al., supra)

Wetting agents, emulsifiers and lubricants, such as sodium laurylsulfate and magnesium stearate, as well as coloring agents, releaseagents, coating agents, sweetening, flavoring and perfuming agents,preservatives and antioxidants can also be present in the compositions.

Examples of pharmaceutically acceptable antioxidants include: (I) watersoluble antioxidants, such as ascorbic acid, cysteine hydrochloride,sodium bisulfate, sodium metabisulfite, sodium sulfite and the like; (2)oil-soluble antioxidants, such as ascorbyl palmitate, butylatedhydroxyanisole (BHA), butylated hydroxytoluene (BHT), lecithin, propylgallate, alpha-tocopherol, and the like; and (3) metal chelating agents,such as citric acid, ethylenediamine tetraacetic acid (EDTA), sorbitol,tartaric acid, phosphoric acid, and the like.

Formulations of the present invention include those suitable for oral,nasal, topical (including buccal and sublingual), rectal, vaginal and/orparenteral administration. The formulations may conveniently bepresented in unit dosage form and may be prepared by any methods wellknown in the art of pharmacy. The amount of active ingredient which canbe combined with a carrier material to produce a single dosage form willvary depending upon the host being treated, the particular mode ofadministration. The amount of active ingredient that can be combinedwith a carrier material to produce a single dosage form will generallybe that amount of the compound which produces a therapeutic effect.Generally, out of one hundred per cent, this amount will range fromabout 1 per cent to about ninety-nine percent of active ingredient,preferably from about 5 per cent to about 70 per cent, most preferablyfrom about 10 per cent to about 30 per cent.

Methods of preparing these formulations or compositions include the stepof bringing into association a compound of the present invention withthe carrier and, optionally, one or more accessory ingredients. Ingeneral, the formulations are prepared by uniformly and intimatelybringing into association a compound of the present invention withliquid carriers, or finely divided solid carriers, or both, and then, ifnecessary, shaping the product.

Formulations of the invention suitable for oral administration may be inthe form of capsules, cachets, pills, tablets, lozenges (using aflavored basis, usually sucrose and acacia or tragacanth), powders,granules, or as a solution or a suspension in an aqueous or non-aqueousliquid, or as an oil-in-water or water-in-oil liquid emulsion, or as anelixir or syrup, or as pastilles (using an inert base, such as gelatinand glycerin, or sucrose and acacia) and/or as mouth washes and thelike, each containing a predetermined amount of a compound of thepresent invention as an active ingredient. A compound of the presentinvention may also be administered as a bolus, electuary or paste.

In solid dosage forms of the invention for oral administration(capsules, tablets, pills, dragees, powders, granules and the like), theactive ingredient is mixed with one or more pharmaceutically acceptablecarriers, such as sodium citrate or dicalcium phosphate, and/or any ofthe following: (1) fillers or extenders, such as starches, lactose,sucrose, glucose, mannitol, and/or silicic acid; (2) binders, such as,for example, carboxymethylcellulose, alginates, gelatin, polyvinylpyrrolidone, sucrose and/or acacia; (3) humectants, such as glycerol;(4) disintegrating agents, such as agar-agar, calcium carbonate, potatoor tapioca starch, alginic acid, certain silicates, and sodiumcarbonate; (5) solution retarding agents, such as paraffin; (6)absorption accelerators, such as quaternary ammonium compounds; (7)wetting agents, such as, for example, cetyl alcohol and glycerolmonostearate; (8) absorbents, such as kaolin and bentonite clay; (9)lubricants, such a talc, calcium stearate, magnesium stearate, solidpolyethylene glycols, sodium lauryl sulfate, and mixtures thereof; and(10) coloring agents. In the case of capsules, tablets and pills, thepharmaceutical compositions may also comprise buffering agents. Solidcompositions of a similar type may also be employed as fillers in softand hard-filled gelatin capsules using such excipients as lactose ormilk sugars, as well as high molecular weight polyethylene glycols andthe like.

A tablet may be made by compression or molding, optionally with one ormore accessory ingredients. Compressed tablets may be prepared usingbinder (for example, gelatin or hydroxypropylmethyl cellulose),lubricant, inert diluent, preservative, disintegrant (for example,sodium starch glycolate or cross-linked sodium carboxymethyl cellulose),surface-active or dispersing agent. Molded tablets may be made bymolding in a suitable machine a mixture of the powdered compoundmoistened with an inert liquid diluent.

The tablets, and other solid dosage forms of the pharmaceuticalcompositions of the present invention, such as dragees, capsules, pillsand granules, may optionally be scored or prepared with coatings andshells, such as enteric coatings and other coatings well known in thepharmaceutical-formulating art. They may also be formulated so as toprovide slow or controlled release of the active ingredient thereinusing, for example, hydroxypropylmethyl cellulose in varying proportionsto provide the desired release profile, other polymer matrices,liposomes and/or microspheres. They may be sterilized by, for example,filtration through a bacteria-retaining filter, or by incorporatingsterilizing agents in the form of sterile solid compositions that can bedissolved in sterile water, or some other sterile injectable mediumimmediately before use. These compositions may also optionally containopacifying agents and may be of a composition that they release theactive ingredient(s) only, or preferentially, in a certain portion ofthe gastrointestinal tract, optionally, in a delayed manner. Examples ofembedding compositions that can be used include polymeric substances andwaxes. The active ingredient can also be in micro-encapsulated form, ifappropriate, with one or more of the above-described excipients.

Liquid dosage forms for oral administration of the compounds of theinvention include pharmaceutically acceptable emulsions, microemulsions,solutions, suspensions, syrups and elixirs. In addition to the activeingredient, the liquid dosage forms may contain inert diluents commonlyused in the art, such as, for example, water or other solvents,solubilizing agents and emulsifiers, such as ethyl alcohol, isopropylalcohol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzylbenzoate, propylene glycol, 1,3-butylene glycol, oils (in particular,cottonseed, groundnut, corn, germ, olive, castor and sesame oils),glycerol, tetrahydrofuryl alcohol, polyethylene glycols and fatty acidesters of sorbitan, and mixtures thereof.

Besides inert diluents, the oral compositions can also include adjuvantssuch as wetting agents, emulsifying and suspending agents, sweetening,flavoring, coloring, perfuming and preservative agents.

Suspensions, in addition to the active compounds, may contain suspendingagents as, for example, ethoxylated isostearyl alcohols, polyoxyethylenesorbitol and sorbitan esters, microcrystalline cellulose, aluminummetahydroxide, bentonite, agar-agar and tragacanth, and mixturesthereof.

It is known that sterols, such as cholesterol, will form complexes withcyclodextrins.

Formulations of the pharmaceutical compositions of the invention forrectal or vaginal administration may be presented as a suppository,which may be prepared by mixing one or more compounds of the inventionwith one or more suitable nonirritating excipients or carrierscomprising, for example, cocoa butter, polyethylene glycol, asuppository wax or a salicylate, and which is solid at room temperature,but liquid at body temperature and, therefore, will melt in the rectumor vaginal cavity and release the active hedgehog antagonist.

Formulations of the present invention which are suitable for vaginaladministration also include pessaries, tampons, creams, gels, pastes,foams or spray formulations containing such carriers as are known in theart to be appropriate.

Dosage forms for the topical or transdermal administration of a compoundof this invention include powders, sprays, ointments, pastes, creams,lotions, gels, solutions, patches and inhalants. The active compound maybe mixed under sterile conditions with a pharmaceutically acceptablecarrier, and with any preservatives, buffers, or propellants that may berequired.

The ointments, pastes, creams and gels may contain, in addition to anactive compound of this invention, excipients, such as animal andvegetable fats, oils, waxes, paraffins, starch, tragacanth, cellulosederivatives, polyethylene glycols, silicones, bentonites, silicic acid,talc and zinc oxide, or mixtures thereof.

Powders and sprays can contain, in addition to a compound of thisinvention, excipients such as lactose, talc, silicic acid, aluminumhydroxide, calcium silicates and polyamide powder, or mixtures of thesesubstances. Sprays can additionally contain customary propellants, suchas chlorofluorohydrocarbons and volatile unsubstituted hydrocarbons,such as butane and propane.

Transdermal patches have the added advantage of providing controlleddelivery of a compound of the present invention to the body. Such dosageforms can be made by dissolving or dispersing the hedgehog antagonistsin the proper medium. Absorption enhancers can also be used to increasethe flux of the hedgehog antagonists across the skin. The rate of suchflux can be controlled by either providing a rate controlling membraneor dispersing the compound in a polymer matrix or gel.

Ophthalmic formulations, eye ointments, powders, solutions and the like,are also contemplated as being within the scope of this invention.

Pharmaceutical compositions of this invention suitable for parenteraladministration comprise one or more compounds of the invention incombination with one or more pharmaceutically acceptable sterileisotonic aqueous or nonaqueous solutions, dispersions, suspensions oremulsions, or sterile powders which may be reconstituted into sterileinjectable solutions or dispersions just prior to use, which may containantioxidants, buffers, bacteriostats, solutes which render theformulation isotonic with the blood of the intended recipient orsuspending or thickening agents.

Examples of suitable aqueous and nonaqueous carriers that may beemployed in the pharmaceutical compositions of the invention includewater, ethanol, polyols (such as glycerol, propylene glycol,polyethylene glycol, and the like), and suitable mixtures thereof,vegetable oils, such as olive oil, and injectable organic esters, suchas ethyl oleate. Proper fluidity can be maintained, for example, by theuse of coating materials, such as lecithin, by the maintenance of therequired particle size in the case of dispersions, and by the use ofsurfactants.

These compositions may also contain adjuvants such as preservatives,wetting agents, emulsifying agents and dispersing agents. Prevention ofthe action of microorganisms may be ensured by the inclusion of variousantibacterial and antifungal agents, for example, paraben,chlorobutanol, phenol sorbic acid, and the like. It may also bedesirable to include isotonic agents, such as sugars, sodium chloride,and the like into the compositions. In addition, prolonged absorption ofthe injectable pharmaceutical form may be brought about by the inclusionof agents that delay absorption such as aluminum monostearate andgelatin.

In some cases, in order to prolong the effect of a drug, it is desirableto slow the absorption of the drug from subcutaneous or intramuscularinjection. This may be accomplished by the use of a liquid suspension ofcrystalline or amorphous material having poor water solubility. The rateof absorption of the drug then depends upon its rate of dissolution,which, in turn, may depend upon crystal size and crystalline form.Alternatively, delayed absorption of a parenterally administered drugform is accomplished by dissolving or suspending the drug in an oilvehicle.

Injectable depot forms are made by forming microencapsule matrices ofthe subject compounds in biodegradable polymers such aspolylactide-polyglycolide. Depending on the ratio of drug to polymer,and the nature of the particular polymer employed, the rate of drugrelease can be controlled. Examples of other biodegradable polymersinclude poly(orthoesters) and poly(anhydrides). Depot injectableformulations are also prepared by entrapping the drug in liposomes ormicroemulsions that are compatible with body tissue.

When the compounds of the present invention are administered aspharmaceuticals, to humans and animals, they call be given per se or asa pharmaceutical composition containing, for example, 0.1 to 99.5% (morepreferably, 0.5 to 90%) of active ingredient in combination with apharmaceutically acceptable carrier.

The addition of the active compound of the invention to animal feed ispreferably accomplished by preparing an appropriate feed premixcontaining the active compound in an effective amount and incorporatingthe premix into the complete ration.

Alternatively, an intermediate concentrate or feed supplement containingthe active ingredient can be blended into the feed. The way in whichsuch feed premixes and complete rations can be prepared and administeredare described in reference books (such as “Applied Animal Nutrition”,W.H. Freedman and CO., San Francisco, U.S.A., 1969 or “Livestock Feedsand Feeding” O and B books, Corvallis, Oreg., U.S.A., 1977).

VI. Synthetic Schemes and Identification of Active Antaeonists

For example, many subject compounds, and congeners thereof, can beprepared readily by employing the cross-coupling technologies of Suzuki,Stille, and the like. These coupling reactions are carried out underrelatively mild conditions and tolerate a wide range of “spectator”functionality.

a. Combinatorial Libraries

The compounds of the present invention, particularly libraries ofvariants having various representative classes of substituents, areamenable to combinatorial chemistry and other parallel synthesis schemes(see, for example, PCT WO 94/08051). The result is that large librariesof related compounds, e.g. a variegated library of compounds representedabove, can be screened rapidly in high throughput assays in orderidentify potential hedgehog antagonist lead compounds, as well as torefine the specificity, toxicity, and/or cytotoxic-kinetic profile of alead compound. For instance, ptc, hedgehog, or smoothened bioactivityassays, such as ay be developed using cells with either a ptcloss-of-function, hedgehog gain-of-function, or smoothenedgain-of-function, can be used to screen a library of the subjectcompounds for those having agonist activity toward ptc or antagonistactivity towards hedgehog or smoothened.

Simply for illustration, a combinatorial library for the purposes of thepresent invention is a mixture of chemically related compounds that maybe screened together for a desired property. The preparation of manyrelated compounds in a single reaction greatly reduces and simplifiesthe number of screening processes that need to be carried out. Screeningfor the appropriate physical properties can be done by conventionalmethods.

Diversity in the library can be created at a variety of differentlevels. For instance, the substrate aryl groups used in thecombinatorial reactions can be diverse in terms of the core aryl moiety,e.g., a variegation in terms of the ring structure, and/or can be variedwith respect to the other substituents.

A variety of techniques are available in the art for generatingcombinatorial libraries of small organic molecules such as the subjecthedgehog antagonists. See, for example, Blondelle et al. (1995) TrendsAnal. Chem. 14:83; the Affymax U.S. Pat. Nos. 5,359,115 and 5,362,899:the Ellman U.S. Pat. No. 5,288,514: the Still et al. PCT publication WO94/08051; the ArQule U.S. Pat. Nos. 5,736,412 and 5,712,171; Chen et al.(1994) JACS 116:2661: Kerr et al. (1993) JACS 115:252; PCT publicationsWO92/10092, WO93/09668 and WO91/07087; and the Lemer et al. PCTpublication WO93/20242). Accordingly, a variety of libraries on theorder of about 100 to 1,000,000 or more diversomers of the subjecthedgehog antagonists can be synthesized and screened for particularactivity or property.

In an exemplary embodiment, a library of candidate hedgehog antagonistsdiversomers can be synthesized utilizing a scheme adapted to thetechniques described in the Still et al, PCT publication WO 94/08051,e.g., being linked to a polymer bead by a hydrolyzable or photolyzablegroup, optionally located at one of the positions of the candidateantagonists or a substituent of a synthetic intermediate. According tothe Still et al. technique, the library is synthesized on a set ofbeads, each bead including a set of tags identifying the particulardiversomer on that bead. The bead library can then be “plated” with ptcloss-of-function, hedgehog gain-of-function, or smoothenedgain-of-function cells for which an hedgehog antagonist is sought. Thediversomers can be released from the bead, e.g., by hydrolysis.

Biological Assays

Lead Compound Discovery/High-Throughput Screening Assay Compounds(10,000) were acquired from Chembridge (San Diego) as DMSO solutions in96-well format. Shh-N (N-terminal fragment of Shh without cholesterolmodification)-conditioned medium was obtained from an HEK 293 cell linestably transfected with Shh-N expression and neomycin resistancecontructs. The Shh-N-producing HEK 293 cells were grown to 80%confluency in DMEM containing 10% (vol/vol) FBS and 400 μg/ml G418. Themedium then was replaced with DMEM containing 2% (vol/vol) FBS, andafter 1 day of growth, the medium was collected and filtered through a0.22-μm membrane. Control medium was obtained from HEK 293 cells.Shh-LIGHT2 cells were then cultured to confluency in 96-well plates andtreated with the small molecules (0.714 μg/ml; ≈2 μM compound in eachwell) in the presence of either Shh-N-conditioned medium or HEK 293control medium (1:25 dilution into DMEM containing 0.5% bovine calfserum). After incubating the treated cells for 30 h at 37° C., cellularfirefly and Renilla luciferase activities were measured (Taipale et al.,Nature 406:1005-1009 (2000)).

Compounds identified in the above assay are depicted below in Schemes1-6.

The compounds of Schemes 5 and 6 inhibit the hedgehog pathway downstreamof smoothened, and thus may effectively inhibit the hedgehog pathway incells that have a smoothened gain-of-function mutation that attenuatesthe activity of compounds that act directly on smoothened or rendersthem ineffective.

All of the publications and references cited above, as well as U.S.Provisional Applications Nos. 60/374,371, filed Apr. 22, 2002,60/405,689, filed Aug. 23, 2002, and 60/415,822, filed Oct. 3, 2002, arehereby incorporated by reference herein.

EQUIVALENTS

Those skilled in the art will recognize, or be able to ascertain usingno more than routine experimentation, many equivalents to the specificembodiments of the invention described herein. Such equivalents areintended to be encompassed by the following claims.

1. A pharmaceutical composition comprising a pharmaceutically acceptableexcipient and a compound having a structure of Formula (I):

wherein, as valence and stability permit, Ar represents a substituted orunsubstituted aryl or heteroaryl ring; X represents, independently foreach occurrence, N or CR′; X₁ represents, independently for eachoccurrence, N or CR″; R′ represents, independently for each occurrence,H, halogen, alkyl, alkenyl, alkynyl, aryl, hydroxyl, alkoxyl, silyloxy,amino, alkylamino, nitro, cyano, thiol, amino, imino, amido, phosphoryl,phosphonate, carboxyl, carboxamide, anhydride, silyl, thioether,alkylsulfonyl, arylsulfonyl, selenoether, acyl, aldehyde, ester,carbamate, or carbonate; R″ represents, independently for eachoccurrence, H, halogen, alkyl, alkenyl, alkynyl, aryl, hydroxyl,alkoxyl, silyloxy, amino, alkylamino, nitro, cyano, thiol, amino, imino,amido, phosphoryl, phosphonate, carboxyl, carboxamide, anhydride, silyl,thioether, alkylsulfonyl, arylsulfonyl, selenoether, acyl, aldehyde,ester, carbamate, or carbonate; R₁ represents a substituted orunsubstituted alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkylalkyl,heterocyclyl, heterocyclyl alkyl, aryl, aralkyl, heteroaryl, orheteroaralkyl; R_(δ) represents from 0-4 substituents on the ring towhich it is attached, independently selected from halogen, alkyl,alkenyl, alkynyl, aryl, hydroxyl, ═O, ═S, alkoxyl, silyloxy, amino,nitro, cyano, thiol, amino, alkylamino, imino, amido, phosphoryl,phosphonate, carboxyl, carboxamide, anhydride, silyl, thioether,alkylsulfonyl, arylsulfony, selenoether, acyl, aldehyde, ester,carbamate and carbonate; and R₉ represents from 0-4 substituents on thering to which it is attached, independently selected from halogen,alkyl, alkenyl, alkynyl, aryl, hydroxyl, alkoxyl, silyloxy, amino,nitro, cyano, thiol, amino, alkylamino, imino, amido, phosphoryl,phosphonate, carboxyl, carboxamide, anhydride, silyl, thioether,alkylsulfonyl, arylsulfonyl, selenoether, acyl, aldehyde, ester,carbamate, and carbonate, or a pharmaceutically acceptable salt thereof.2. A composition of claim 1, wherein Ar represents a substituted orunsubstituted phenyl ring, X and X₁ represent N for all occurrences, R₁represents alkyl, heteroaralkyl, or aralkyl, R₈ represents 0substituents, and R₉ represents two methyl groups separately located onthe two unsubstituted carbon atoms of the ring to which it is attached.3. A pharmaceutical composition comprising a compound having thestructure

or a pharmaceutically acceptable salt thereof.
 4. A pharmaceuticalcomposition comprising a pharmaceutically acceptable excipient and acompound having a structure of Formula II:

wherein, as valence and stability permit, A represents a pyridyl ordihydropyridyl ring; Ar represents a substituted or unsubstituted arylor heteroaryl ring; and R₉ represents from 0-4 substituents on the ringto which it is attached, independently selected from halogen, alkyl,alkenyl, alkynyl, aryl, hydroxyl, alkoxyl, silyloxy, amino, nitro,cyano, thiol, amino, alkylamino, imino, amido, phosphoryl, phosphonate,carboxyl, carboxamide, anhydride, silyl, thioether, alkylsulfonyl,arylsulfonyl, selenoether, acyl, aldehyde, ester, carbamate, andcarbonate, or a pharmaceutically acceptable salt thereof.
 5. Apharmaceutical composition comprising a pharmaceutically acceptableexcipient and a compound having a structure of Formula III:

wherein, independently for each occurrence, A represents a pyridyl ordihydropyridyl ring; Ar represents a substituted or unsubstituted arylor heteroaryl ring; and R₂ represents a thioether, preferablysubstituted with an amido, ester, carboxyl, or acyl; R₃ represents H,alkyl, alkenyl, alkynyl, or cycloalkyl; and R₄ represents a substituentselected from H, halogen, alkyl, alkenyl, alkynyl, aryl, hydroxyl,alkoxyl, silyloxy, amino, nitro, cyano, thiol, amino, alkylamino, imino,amido, phosphoryl, phosphonate, carboxyl, carboxamide, anhydride, silyl,thioether, alkylsulfonyl, arylsulfonyl, selenoether, acyl, aldehyde,ester, carbamate, and carbonate, or a pharmaceutically acceptable saltthereof.
 6. A pharmaceutical composition comprising a pharmaceuticallyacceptable excipient and a compound having a structure of Formula IV:

wherein, as valence and stability permit, Ar represents a substituted orunsubstituted aryl or heteroaryl ring; W represents C═O, C═S, or SO₂;Har represents a substituted or unsubstituted heteroaryl ring; Htrepresents OR or NR′″₂; R represents H or lower alkyl; and R′″represents H, lower alkyl, cycloalkyl, cycloalkylalkyl, heterocyclyl,heterocyclyl alkyl, aryl, aralkyl, heteroaryl, or heteroaralkyl, or apharmaceutically acceptable salt thereof.
 7. A composition of claim 6,wherein Ar represents a substituted or unsubstituted phenyl ring, andHar represents a substituted or unsubstituted 2-pyridyl ring.
 8. Apharmaceutical composition comprising a pharmaceutically acceptableexcipient and a compound having a structure of Formula V:

wherein, as valence and stability permit, Ar represents a substituted orunsubstituted aryl or heteroaryl ring, preferably a substituted orunsubstituted phenyl ring, such as an ortho-hydroxyphenyl groupoptionally substituted with one or more halogen, hydroxy, alkoxy, amino,alkylamino, alkyl, thiol, alkylthio, ester, amido, carboxyl, nitro, orcyano substituents; Y represents OH or NR2; Z represents O or S; Rrepresents H or lower alkyl; and R₅ represents substituted orunsubstituted lower alkyl, or a pharmaceutically acceptable saltthereof.
 9. A composition of claim 8, wherein R₅ represents atrihalomethyl group, Y represents NH2, and Ar represents an optionallyfurther substituted 2-hydroxyphenyl substituent.
 10. A pharmaceuticalcomposition comprising a pharmaceutically acceptable excipient and acompound having a structure of Formula VI:

wherein, as valence and stability permit, Ar, independently for eachoccurrence, represents a substituted or unsubstituted aryl or heteroarylring; W represents C═O, C═S, or SO₂; X represents, independently foreach occurrence, N or CR′; X₁ represents, independently for eachoccurrence, N or CR″; R represents H or lower alkyl; and R′ represents,independently for each occurrence, H, halogen, allcyl, alkenyl, alkynyl,aryl, hydroxyl, alkoxyl, silyloxy, amino, alkylamino, nitro, cyano,thiol, amino, imino, amido, phosphoryl, phosphonate, carboxyl,carboxamide, anhydride, silyl, thioether, alkylsulfonyl, arylsulfonyl,selenoether, acyl, aldehyde, ester, carbamate, or carbonate; R″represents, independently for each occurrence, H, halogen, alkyl,alkenyl, alkynyl, aryl, hydroxyl, alkoxyl, silyloxy, amino, alkylamino,nitro, cyano, thiol, amino, imino, amido, phosphoryl, phosphonate,carboxyl, carboxamide, anhydride, silyl, thioether, alkylsulfonyl,arylsulfonyl, selenoether, acyl, aldehyde, ester, carbamate, orcarbonate; and R₉ represents from 0-4 substituents on the ring to whichit is attached, independently selected from halogen, alkyl, alkenyl,alkynyl, aryl, hydroxyl, alkoxyl, silyloxy, amino, nitro, cyano, thiol,amino, alkylamino, imino, amido, phosphoryl, phosphonate, carboxyl,carboxamide, anhydride, silyl, thioether, alkylsulfonyl, arylsulfonyl,selenoether, acyl, aldehyde, ester, carbamate, and carbonate, or apharmaceutically acceptable salt thereof.
 11. A composition of claim 10,wherein Ar represents a substituted or unsubstituted phenyl ring, Xrepresents N, X₁ represents CR, and R₉ is absent or represents 1-4substituents selected from halogen, alkyl, cyano, and nitro.
 12. Apharmaceutical composition comprising a pharmaceutically acceptableexcipient and a compound having a structure of Formula VII:

wherein, as valence and stability permit, X and Z, independently,represent —N(R₇)—, —O—, —S—, —(R₇)N—N(R₇)—, —ON(R₇)—, or a direct bond;Y represents —C(═O)—, —C(═S)—, —C(═NR₇)—, SO₂, or SO; A represents O, S,or NR₇; G represents a cycloalkyl, heterocyclyl, aryl, or heteroarylring fused to the ring to which it is attached; Ar represents asubstituted or unsubstituted aryl or heteroaryl ring; R₁ represents H orsubstituted or unsubstituted alkyl, alkenyl, alkynyl, aryl, heteroaryl,heterocyclyl, or cycloalkyl, including polycyclic groups; R₂ representsfrom 0-4 substituents on the ring to which it is attached, such ashalogen, lower alkyl, lower alkenyl, aryl, heteroaryl, carbonyl group,thiocarbonyl, ketone, aldehyde, amino, acylamino, amido, amidino, cyano,nitro, azido, sulfonyl, sulfoxido, sulfate, sulfonate, sulfamoyl,sulfonamido, phosphoryl, phosphonate, phosphinate, J-R₈, J-OH, J-loweralkyl, J-lower alkenyl, J-R₈, J-SH, J-NH₂, protected forms of the above,or any two R₂, when occurring more than once in a cyclic or polycyclicstructure, can be taken together form a 4- to 8-membered cycloalkyl,aryl, or heteroaryl; R₇, independently for each occurrence, represents Hor substituted or unsubstituted lower alkyl, J-cycloalkyl,J-heterocyclyl, J-aryl, or J-heteroaryl; R₈, independently for eachoccurrence, represents H or substituted or unsubstituted lower alkyl,cycloalkyl, heterocyclyl, aryl, or heteroaryl; and J represents,independently for each occurrence, a chain having from 0-8 unitsselected from CK₂, NK, O, and S, wherein K represents, independently foreach occurrence, H or lower alkyl.
 13. The composition of claim 1,wherein the compound inhibits ptc loss-of-function, hedgehoggain-of-function, or smoothened gain-of-function mediated signaltransduction with an IC₅₀ of 1 μM or less.
 14. A method of inhibitingthe hedgehog pathway in a cell, comprising contacting the cell with acomposition of claim
 1. 15. The method of claim 14, wherein the cell hasa phenotype of ptc loss-of-function, hedgehog gain-of-function, orsmoothened gain-of-function.
 16. The method of claim 14, wherein thecell is contacted with the hedgehog antagonist in vivo.
 17. The methodof claim 14, wherein the cell is contacted with the hedgehog antagonistin vitro.
 18. The method of claim 14, wherein the compound isadministered to an animal as part of a therapeutic or cosmeticapplication.
 19. The method of claim 18, wherein the therapeutic orcosmetic application is selected from regulation of neural tissues, boneand cartilage formation and repair, regulation of spermatogenesis,regulation of smooth muscle, regulation of lung, liver and other organsarising from the primitive gut, regulation of hematopoietic function,and regulation of skin and hair growth.
 20. A method for inhibitingunwanted proliferation of a cell, comprising contacting the cell with acomposition of claim
 1. 21. The method of claim 20, wherein the cell isa cancer cell.
 22. The method of claim 21, wherein the cell is a cancercell from a cancer selected from medulloblastoma, rhabdomyosarcoma,endodermally or gut-derived cancers (including small cell lung cancer,esophageal cancer, gastric (stomach) cancer, biliary cancer, andpancreatic cancer), urogenital cancers (including prostate cancer andbladder cancer), ovarian cancer, and uterine cancer.
 23. A method fortreating or preventing a carcinoma, comprising administering acomposition of claim 1 to a patient in an amount sufficient to inhibitprogression of the carcinoma.
 24. The method of claim 23, wherein thecarcinoma is a basal cell carcinoma.
 25. A method of inhibiting thehedgehog pathway in a cell, comprising contacting the cell with acompound that inhibits activation of the hedgehog pathway downstream ofsmoothened.